CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. Nos. 62/805,743 and 62/896,343, filed on Feb. 14, 2019 and Sep. 5, 2019, respectively, the disclosures of which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 14, 2020, is named 2012383.txt and is 53,248 bytes in size.

TECHNICAL FIELD

This disclosure describes production of omega-3 docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and/or eicosapentaenoic acid (EPA) at elevated levels in seeds of transgenic Brassica plants. Seeds and oils obtained from such seeds that have higher levels of DHA, DPA, and/or EPA have certain beneficial effects in their fatty acid profiles, such as reductions in the levels of saturated fatty acids (e.g., stearic acid).

BACKGROUND

Aquaculture is a fast-growing industry where shrimp and various fish such as salmon, tilapia, halibut, carp, channel catfish, trout, sea bream and sea bass can be grown under controlled conditions. Typically, farmed fish are fed formulations containing fish oil and/or omega-3 long chain polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to ensure that the farmed fish can deliver the health benefits of the omega-3 fish oils to consumers. Aquaculture production is expected to grow several times in the coming decades, while fishmeal and fish oil production will be about constant. As such, there is a need for alternative sources of omega-3 long chain polyunsaturated fatty acids.

SUMMARY

This disclosure is based, at least in part, on the discovery that Brassica plants can be produced in which the seeds from these plants yield oils with elevated levels of long chain polyunsaturated fatty acids such as omega-3 docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and/or eicosapentaenoic acid (EPA). For example, the Brassica plants, or parts thereof, described herein can have higher levels of EPA, higher levels of DPA, higher levels of DHA, higher levels of EPA and DHA, higher levels of DHA and DPA, higher levels of DPA and EPA, or higher levels of EPA, DHA, and DPA.

In one aspect, this disclosure provides a Brassica plant or a part thereof comprising one or more exogenous polynucleotides heritably integrated into its genome, the exogenous polynucleotides comprising one or more expression cassettes having nucleotide sequences encoding one or more d12DES, one or more d6Elo, one or more d6Des, one or more d5Des, one or more d5Elo, one or more d4Des, and/or one or more o3Des. The plant can be the result of crossing a first parental Brassica plant that comprises the one or more exogenous polynucleotides with a second parental Brassica plant. The Brassica plant produces in its seeds a greater amount of one or more polyunsaturated fatty acids selected from the group consisting of EPA, DPA, and DHA than the first parental Brassica plant and/or the second parental Brassica plant. Apart of a Brassica plant includes any parts derived from a plant, including cells, tissues, roots, stems, leaves, non-living harvest material, silage, seeds, seed meals and pollen.

In another aspect, provided is a method of producing a Brassica plant or a part thereof, the method comprising crossing a first Brassica parent plant producing one or more polyunsaturated fatty acids selected from the group consisting of EPA, DPA, and DHA in its seeds with a second Brassica parent plant to produce progeny plants producing one or more of EPA, DPA, and DHA. In some embodiments, the progeny Brassica plant produces greater levels of DHA and/or EPA than the first or second Brassica parent. In some embodiments, the first Brassica plant has one or more exogenous polynucleotides (e.g., T-DNAs) heritably integrated into its genome. In some embodiments, the second Brassica plant contributes at least one genomic sequence that confers in part, or in whole, the higher amount of one or more of EPA, DPA, and DHA. This genomic sequence can be referred to as a quantitative trait locus (QTL). The genomic sequence from the second parent can be all or a part of the genomic sequence selected from the group consisting of a) the genomic sequence on chromosome N1 between nucleotide positions 8879780 and 11922690; b) the genomic sequence on chromosome N1 between nucleotide positions 22823086 and 24045492; and c) the genomic sequence on chromosome N6 between nucleotide positions 19156645 and 20846412.

This disclosure also provides a Brassica plant or a part thereof that includes (i) one or more exogenous polynucleotides (e.g., T-DNAs) heritably integrated into its genome, the exogenous polynucleotides comprising one or more expression cassettes having nucleotide sequences encoding one or more desaturases and/or one or more elongases; and (ii) all or part of at least one genomic sequence of aB. napus parent genome that confers a higher amount of one or more polyunsaturated fatty acids selected from the group consisting of EPA, DPA, and DHA, wherein the genome sequence is selected from the group consisting of: a) the genomic sequence on chromosome N1 between nucleotide positions 8,879,780 and 11,922,690; b) the genomic sequence on chromosome N1 between nucleotide positions 22,823,086 and 24,045,492; and c) the genomic sequence on chromosome N6 between nucleotide positions 19,156,645 and 20,846,412; wherein seeds of the Brassica plant have a greater amount of one or more polyunsaturated fatty acids selected from the group consisting of EPA, DPA, and DHA than seeds of a control Brassica plant lacking (i) and/or (ii). In some embodiments, the genomic sequence comprises all or part of the genomic sequence on chromosome N1 between nucleotide positions 8,879,780 and 11,922,690 and the genomic sequence on chromosome N1 between nucleotide positions 22,823,086 and 24,045,492. In some embodiments, the genomic sequence comprises all or part of the genomic sequence on chromosome N1 between nucleotide positions 8,879,780 and 11,922,690 and the genomic sequence on chromosome N6 between nucleotide positions 19,156,645 and 20,846,412. In some embodiments, the genomic sequence comprises all or part of the genomic sequence on chromosome N1 between nucleotide positions 22,823,086 and 24,045,492 and the genomic sequence on chromosome N6 between nucleotide positions 19,156,645 and 20,846,412. In some embodiments, the genomic sequence can include from 25 to 50, 25 to 100, 50 to 200, 100 to 500, 250 to 1,000, 500 to 5,000, 2,000 to 10,000, 5,000 to 20,000, 10,000 to 100,000, 50,000 to 400,000, 25,000 to 1,000,000, 100,000 to 1,000,000, 200,000 to 1,000,000, or 500 to 1,000,000 contiguous nucleotides of the genomic sequence of the B. napus parent genome.

The genomic sequence on chromosome N1 between nucleotide positions 8,879,780 and 11,922,690 can include a single nucleotide polymorphism (SNP) at a position selected from the group consisting of 8,952,616, 9,040,901, 9,046,609, 9,048,617, 9,136,686, 9,143,608, 9,248,592, 9,347,120, 9,352,326, 9,454,361, 9,549,523, 9,641,936, 9,652,028, 9,794,198, 9,847,417, 9,921,975, 9,952,792, 10,052,015, 10,402,684, 10,425,211, 10,558,464, 10,613,015, 10,659,284, 10,706,805, 10,748,492, 10,852,010, 11,007,740, 11,047,958, 11,150,929, 11,269,217, 11,343,118, 11,455,979, 11,565,970, 11,659,776, 11,726,807, 11,850,103, and 11,956,477. In some embodiments, the genomic sequence includes 5, 10, 15, 20, 30, 35, or 40 SNPs at different positions selected from the group consisting of 8,952,616, 9,040,901, 9,046,609, 9,048,617, 9,136,686, 9,143,608, 9,248,592, 9,347,120, 9,352,326, 9,454,361, 9,549,523, 9,641,936, 9,652,028, 9,794,198, 9,847,417, 9,921,975, 9,952,792, 10,052,015, 10,402,684, 10,425,211, 10,558,464, 10,613,015, 10,659,284, 10,706,805, 10,748,492, 10,852,010, 11,007,740, 11,047,958, 11,150,929, 11,269,217, 11,343,118, 11,455,979, 11,565,970, 11,659,776, 11,726,807, 11,850,103, and 11,956,477.

The genomic sequence on chromosome N1 between nucleotide positions 8,879,780 and 11,922,690 can include at least one SNP at a position selected from the group consisting of 9,136,686, 9,641,936, 10,613,015, 9,040,901, 9,048,617, 9,352,326, 9,921,975, and 10,706,805. In some embodiments, the genomic sequence includes 2, 3, 4, 5, 6, 7, or 8 SNPs at different positions selected from the group consisting of U.S. Pat. Nos. 9,136,686, 9,641,936, 10,613,015, 9,040,901, 9,048,617, 9,352,326, 9,921,975, and 10,706,805.

The genomic sequence on chromosome N1 between nucleotide positions 22,823,086 and 24,045,492 can include a SNP at a position selected from the group consisting of 22,823,086, 22,880,595, 22,902,670, 22,949,738, 23,011,207, 23,044,228, 23,099,592, 23,176,771, 23,201,595, 23,257,618, 23,302,268, 23,367,822, 23,380,089, 23,457,696, 23,520,607, 23,552,773, 23,598,941, 23,670,623, 23,682,848, 23,745,365, 23,792,572, 23,855,829, 23,910,029, 23,947,522, 24,021,883. In some embodiments, the genomic sequence includes 5, 10, 15, 20, 25, or 30 SNPs at different positions selected from the group consisting of 22,823,086, 22,880,595, 22,902,670, 22,949,738, 23,011,207, 23,044,228, 23,099,592, 23,176,771, 23,201,595, 23,257,618, 23,302,268, 23,367,822, 23,380,089, 23,457,696, 23,520,607, 23,552,773, 23,598,941, 23,670,623, 23,682,848, 23,745,365, 23,792,572, 23,855,829, 23,910,029, 23,947,522, 24,021,883, 24,056,999.

The genomic sequence on chromosome N1 between nucleotide positions between nucleotide positions 22,823,086 and 24,045,492 can include a SNP at a position selected from the group consisting of 23,089,542, 23,089,635, 23,090,743, 23,090,785, 23,091,367, 23,092,042, 23,150,402, 23,150,595, 23,155,220, 23,155,766, 23,314,197, 23,318,357, 23,343,089, 23,679,276, 23,679,287, 23,679,396, 23,886,929, 23,925,895, 23,963,309, 24,029,270, 24,029,279, 24,029,294. In some embodiments, the genomic sequence includes 5, 10, 15, 20, or 25 SNPs at different positions selected from the group consisting of 23,089,542, 23,089,635, 23,090,743, 23,090,785, 23,091,367, 23,092,042, 23,150,402, 23,150,595, 23,155,220, 23,155,766, 23,314,197, 23,318,357, 23,343,089, 23,679,276, 23,679,287, 23,679,396, 23,886,929, 23,925,895, 23,963,309, 24,029,270, 24,029,279, 24,029,294.

The genomic sequence on chromosome N6 between nucleotide positions 19,156,645 and 20,846,412 can include a SNP at a position selected from the group consisting of 19,156,645, 19,199,109, 19,325,186, 19,402,086, 19,513,420, 19,583,431, 19,601,021, 19,706,563, 19,800,643, 19,906,666, 20,000,119, 20,095,002, 20,205,211, 20,300,571, 20,406,148, 20,407,023, 20,505,840, 20,601,198, 20,631,917, and 20,702,631. In some embodiments, the genomic sequence includes at least 5, 10, 15, 20, 25, 30 SNPs at different positions selected from the group consisting of 19,156,645, 19,199,109, 19,325,186, 19,402,086, 19,513,420, 19,583,431, 19,601,021, 19,706,563, 19,800,643, 19,906,666, 20,000,119, 20,095,002, 20,205,211, 20,300,571, 20,406,148, 20,407,023, 20,505,840, 20,601,198, 20,631,917, and 20,702,631.

The genomic sequence on chromosome N6 between nucleotide positions 19,156,645 and 20,846,412 can include a SNP at a position selected from the group consisting of 19,336,744, 19,336,819, 19,337,615, 19,350,156, 19,353,584, 19,353,648, 19,353,749, 19,476,836, 19,783,834, 19,784,007, 19,784,367, 19,784,633, 19,784,672, 19,784,688, 19,784,733, 19,800,525, 20,191,826, 20,300,548, 20,375,643, 20,766,637, 20,769,461, 20,770,769, 20,823,998, 20,825,959, 20,826,301, 20,827,570, 20,827,573. In some embodiments, the genomic sequence includes at least 5, 10, 15, 20, 25, 30, 35, or 40 SNPs at different positions selected from the group consisting of 19,336,744, 19,336,819, 19,337,615, 19,350,156, 19,353,584, 19,353,648, 19,353,749, 19,476,836, 19,783,834, 19,784,007, 19,784,367, 19,784,633, 19,784,672, 19,784,688, 19,784,733, 19,800,525, 20,191,826, 20,300,548, 20,375,643, 20,766,637, 20,769,461, 20,770,769, 20,823,998, 20,825,959, 20,826,301, 20,827,570, 20,827,573.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. he materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of the different enzymatic activities leading to the production of ARA, EPA and DHA.

FIG. 2 shows the distribution of EPA, DPA and DHA contents from 279 Brassica accessions. Arrow shows the average content from the PUFA donor line, Kumily LBFLFK.

FIG. 3 is a Manhattan plot showing two genomic blocks on N01 for EPA.

FIG. 4 is a Manhattan plot showing the genomic block on N01 for DPA.

FIG. 5 is a Manhattan plot showing the two genomic blocks on N01 for DHA.

FIG. 6 is a Manhattan plot showing the genomic block on N06 for EPA.

DETAILED DESCRIPTION

As described herein, Brassica plants can be produced in which the seeds from these plants yield oils with elevated levels of long chain polyunsaturated fatty acids. The term “polyunsaturated fatty acids (PUFA)” as used herein refers to fatty acids comprising at least two (e.g., at least three, four, five or six) double bonds in a fatty acid chain that is, for example, from 18 to 24 carbon atoms in length. In some embodiments, the term relates to very long chain PUFA (VLC-PUFA) having from 20 to 24 carbon atoms in the fatty acid chain. PUFAs can be, for example, dihomo-gamma linolenic acid (DHGLA, 20:3 (8,11,14)), arachidonic acid (ARA, 20:4 (5,8,11,14)), EPA (20:5 (5,8,11,14,17)), docosapentaenoic acid (DPA, 22:5 (4,7,10,13,16)), DHA (22:6 (4,7,10,13,16,19)), and/or eicosatetraenoic acid (ETA, 20:4 (8,11,14,17)). In some embodiments, seeds of Brassica plants provided herein can produce higher levels of EPA, higher levels of DPA, higher levels of DHA, higher levels of ARA, higher levels of EPA and DHA, higher levels of DHA and DPA, higher levels of DPA and EPA, higher levels of ARA, EPA, and DHA, or higher levels of EPA, DHA, and DPA.

In some embodiments, the Brassica plants or parts thereof can produce one or more intermediates of VLC-PUFA which occur during synthesis. Such intermediates can be formed from substrates by one or more activities of a desaturase, keto-acyl-CoA-synthase, keto-acyl-CoA-reductase, dehydratase, or enoyl-CoA-reductase polypeptide. In some embodiments, substrates can be linoleic acid (LA, 18:2 (9,12)), gamma linolenic acid (GLA 18:3 (6,9,12)), DHGLA, ARA, eicosadienoic acid 20:2 (11,14), ETA, or EPA.

In some embodiments, a Brassica plant or part thereof provided herein can be produced by crossing a first Brassica plant with a second Brassica plant and selecting progeny. In some embodiments, the first Brassica plant can include one or more expression cassettes comprising at least one polynucleotide sequence encoding one or more desaturases and/or one or more elongases. In some embodiments, the second Brassica plant contributes at least one genomic sequence that is in part or in whole responsible for the higher levels of DHA and/or EPA.

The term “polynucleotide” according to the present disclosure refers to a deoxyribonucleic acid or ribonucleic acid. Unless stated otherwise, “polynucleotide” herein refers to a single strand of a DNA polynucleotide or to a double stranded DNA polynucleotide. As used herein, the terms nucleotide/polynucleotide and nucleotide sequence/polynucleotide sequence are used interchangeably, and such terms encompass both double stranded and single stranded nucleic acids.

The term “desaturase” encompasses all enzymatic activities and enzymes catalyzing the desaturation of fatty acids with different lengths and numbers of unsaturated carbon atom double bonds. For example, a desaturase can be a delta 4 (d4)-desaturase that catalyzes the dehydrogenation of the 4th and 5th carbon atom; a delta 5 (d5)-desaturase catalyzing the dehydrogenation of the 5th and 6th carbon atom; a delta 6 (d6)-desaturase catalyzing the dehydrogenation of the 6th and 7th carbon atom; a delta 8 (d8)-desaturase catalyzing the dehydrogenation of the 8th and 9th carbon atom; a delta 9 (d9)-desaturase catalyzing the dehydrogenation of the 9th and 10th carbon atom; a delta 12 (d12)-desaturase catalyzing the dehydrogenation of the 12th and 13th carbon atom; or a delta 15 (d15)-desaturase catalyzing the dehydrogenation of the 15th and 16th carbon atom.

The terms “elongase” encompasses all enzymatic activities and enzymes catalyzing the elongation of fatty acids with different lengths and numbers of unsaturated carbon atom double bonds. In some embodiments, the term “elongase” refers to the activity of an elongase that introduces two carbon molecules into the carbon chain of a fatty acid.

In some embodiments, the one or more expression cassettes can have polynucleotide sequences encoding one or more d5Des, one or more d6Elo, one or more d5Des, one or more o3Des, one or more d5Elo and one or more d4Des, for example, for at least one CoA-dependent D4Des and one phospholipid-dependent d4Des. In some embodiments, one or more d12Des also are encoded.

In some embodiments, the one or more expression cassettes can have polynucleotide sequences encoding at least two d6Des, at least two d6Elo, and/or at least two o3Des. In some embodiments, the one or more expression cassettes also can encode at least one CoA-dependent d4Des and at least one phospholipid dependent d4Des.

Polynucleotides encoding polypeptides that exhibit delta-6-elongase activity have been described, for example, in WO2001/059128, WO2004/087902, WO2005/012316, and WO 2015/089587, which are incorporated herein in their entirety. Non-limiting exemplary delta-6-elongases include those from Physcomitrella patens and Pyramimonas cordata.

Polynucleotides encoding polypeptides which exhibit delta-5-desaturase (d5Des) activity have been described, for example, in WO2002/026946, WO2003/093482, and WO 2015/089587, which are incorporated herein in their entirety. Non-limiting exemplary delta-5-desaturases include those from Thraustochytrium sp., Pavlova salina, and Pyramimonas cordata.

Polynucleotides encoding polypeptides which exhibit delta-6-desaturase activity have been described in WO2005/012316, WO2005/083093, WO2006/008099 and WO2006/069710, and WO 2015/089587, which are incorporated herein in their entirety. Non-limiting exemplary delta-6-desaturases include those from Ostreococcus tauri, Micromonas pusilla, and Osreococcus lucimarinus.

Polynucleotides encoding polypeptides which exhibit delta-5-elongase activity have been described in WO2005/012316, WO2005/007845, WO2007/096387, WO2006/069710, and WO 2015/089587, which are incorporated herein in their entirety. Non-limiting exemplary delta-5-elongases include those from Ostreococcus tauri and Pyramimonas cordata.

Polynucleotides encoding polypeptides which exhibit delta-12-desaturase activity have been described for example in WO2006100241 and WO 2015/089587, which are incorporated herein in their entirety. Non-limiting exemplary delta-12-desaturases include those from Phytophthora sojae and Lachancea kluyveri.

Polynucleotides encoding polypeptides which exhibit delta-4-desaturase (d4Des) activity have been described for example in WO2004/090123, WO2002026946, WO2003078639, WO2005007845, and WO 2015/089587, which are incorporated herein in their entirety. Non-limiting exemplary delta-4-desaturases include those from Euglena gracilis, Thraustochytrium sp., Pavlova lutheri, and Pavlova salina. See, e.g., delta-4 desaturase “PIDES 1” and FIGS. 3a-3d of WO2003078639 and FIGS. 3a, 3b of WO2005007845, respectively.

Polynucleotides encoding polypeptides which exhibit omega 3-desaturase (o3Des) activity have been described for example in WO2008/022963, WO2005012316, WO2005083053, and WO 2015/089587, which are incorporated herein in their entirety. Non-limiting exemplary omega-3-desaturases include those from Phytium irregular, Phytophthora infestans, and Pichia pastoris.

Polynucleotides encoding polypeptides which exhibit delta-15-desaturase activity have been described for example in WO2010/066703, which is incorporated herein in its entirety. Non-limiting exemplary delta-15 destaurases include the delta-15 desaturase from Cochliobolus heterostrophus C5.

Additional polynucleotides that encode polypeptides having desaturase or elongase activities as specified above can be obtained from various organisms, including but not limited to, organisms of genus Ostreococcus, Thraustochytrium, Euglena, Thalassiosira, Phytophthora, Phytium, Cochliobolus, or Physcomitrella. Orthologs, paralogs or other homologs having suitable desaturase or elongase activities may be identified from other species. In some embodiments, such orthologs, paralogs, or homologs are obtained from plants such as algae, for example Isochrysis, Mantoniella, or Crypthecodinium, algae/diatoms such as Phaeodactylum, mosses such as Ceratodon, or higher plants such as the Primulaceae such as Aleuritia, Calendula stellata, Osteospermum spinescens or Osteospermum hyoseroides, microorganisms such as fungi, such as Aspergillus, Entomophthora, Mucor or Mortierella, bacteria such as Shewanella, yeasts or animals. Non-limiting exemplary animals are nematodes such as Caenorhabditis, insects or vertebrates. Among the vertebrates, the nucleic acid molecules may, in some embodiments, be derived from Euteleostomi, Actinopterygii; Neopterygii; Teleostei; Euteleostei, Protacanthopterygii, Salmonformes; Salmonidae or Oncorhynchus, such as from the order of the Salmoniformes, such as the family of the Salmonidae, such as the genus Salmo, for example from the genera and species Oncorhynchus mykiss, Trutta trutta or Salmo trutta fario. Moreover, the nucleic acid molecules may be obtained from the diatoms such as the genera Thalassiosira or Phaeodactylum.

The term “polynucleotide” as used herein further encompasses variants, muteins or derivatives of the aforementioned specific polynucleotides that are suitable for use in embodiments of the present disclosure.

Nucleic acid variants or derivatives according to the disclosure are polynucleotides which differ from a given reference polynucleotide by at least one nucleotide substitution, addition and/or deletion. If the reference polynucleotide codes for a protein, the function of this protein is conserved in the variant or derivative polynucleotide, such that a variant nucleic acid sequence shall still encode a polypeptide having a desaturase or elongase activity as specified above. Variants or derivatives also encompass polynucleotides comprising a nucleic acid sequence which is capable of hybridizing to the aforementioned specific nucleic acid sequences, for example, under stringent hybridization conditions. These stringent conditions are known in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. A non-limiting example for stringent hybridization conditions are hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at approximately 45° C., followed by one or more wash steps in 0.2×SSC, 0.1% SDS at 50 to 65° C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. For example, under “standard hybridization conditions” the temperature differs depending on the type of nucleic acid between 42° C. and 58° C. in aqueous buffer with a concentration of 0.1 to 5′ SSC (pH 7.2). If organic solvent is present in the abovementioned buffer, for example 50% formamide, the temperature under standard conditions is approximately 42° C. The hybridization conditions for DNA: DNA hybrids are, for example, 0.1×SSC and 20° C. to 45° C., such between 30° C. and 45° C. The hybridization conditions for DNA:RNA hybrids are, for example, 0.1×SSC and 30° C. to 55° C., such as between 45° C. and 55° C. The abovementioned hybridization temperatures are determined for example for a nucleic acid with approximately 100 bp (=base pairs) in length and a G+C content of 50% in the absence of formamide. The skilled worker knows how to determine the hybridization conditions required by referring to textbooks such as the textbook mentioned above, or the following textbooks: Sambrook et al., “Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, “Nucleic Acids Hybridization: A Practical Approach”, IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, “Essential Molecular Biology: A Practical Approach”, IRL Press at Oxford University Press, Oxford. Alternatively, polynucleotide variants are obtainable by PCR-based techniques such as mixed oligonucleotide primer-based amplification of DNA, i.e. using degenerated primers against conserved domains of the polypeptides of the present disclosure. Conserved domains of a polypeptide suitable for use in embodiments of the present disclosure may be identified by a sequence comparison of the nucleic acid sequences of the polynucleotides or the amino acid sequences of the polypeptides of the present disclosure. Oligonucleotides suitable as PCR primers as well as suitable PCR conditions are described in the accompanying Examples. As a template, DNA or cDNA from bacteria, fungi, plants or animals may be used. Further, variants include polynucleotides comprising nucleic acid sequences which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the nucleic acid coding sequences shown in any one of the T-DNA sequences. Of course, the variants must retain the function of the respective enzyme, i.e., a variant of a delta-4-desaturase must retain delta-4-desaturase activity.

The percent identity values are, in some embodiments, calculated over the entire amino acid or nucleic acid sequence region. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In some embodiments, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (Needleman 1970, J. Mol. Biol. (48):444-453) which has been incorporated into the needle program in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice, P., Longden,I., and Bleasby,A, Trends in Genetics 16(6), 276-277, 2000), a BLOSUM62 scoring matrix, and a gap opening penalty of 10 and a gap extension penalty of 0.5. Non-limiting example of parameters to be used for aligning two amino acid sequences using the needle program are the default parameters, including the EBLOSUM62 scoring matrix, a gap opening penalty of 10 and a gap extension penalty of 0.5. In yet another embodiment, the percent identity between two nucleotide sequences is determined using the needle program in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice, P., Longden, I., and Bleasby, A, Trends in Genetics 16(6), 276-277, 2000), using the EDNAFULL scoring matrix and a gap opening penalty of 10 and a gap extension penalty of 0.5. A non-limiting example of parameters to be used in conjunction for aligning two nucleic acid sequences using the needle program are the default parameters, including the EDNAFULL scoring matrix, a gap opening penalty of 10 and a gap extension penalty of 0.5. The nucleic acid and protein sequences of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLAST series of programs (version 2.2) of Altschul et al. (Altschul 1990, J. Mol. Biol. 215:403-10). BLAST using desaturase and elongase nucleic acid sequences of the disclosure as query sequence can be performed with the BLASTn, BLASTx or the tBLASTx program using default parameters to obtain either nucleotide sequences (BLASTn, tBLASTx) or amino acid sequences (BLASTx) homologous to desaturase and elongase sequences of the disclosure. BLAST using desaturase and elongase protein sequences of the disclosure as query sequence can be performed with the BLASTp or the tBLASTn program using default parameters to obtain either amino acid sequences (BLASTp) or nucleic acid sequences (tBLASTn) homologous to desaturase and elongase sequences ofthe disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST using default parameters can be utilized as described in Altschul et al. (Altschul 1997, Nucleic Acids Res. 25(17):3389-3402).

The variant polynucleotides or fragments referred to above, in some embodiments, encode polypeptides retaining desaturase or elongase activity to a significant extent, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the desaturase or elongase activity exhibited by any of the polypeptide comprised in any of the T-DNAs disclosed herein.

Further enzymes that may be used in embodiments of the present disclosure include, but are not limited to, acyltransferases and transacylases (see, for example, WO 2011161093), such as, for example, lysophosphatidic acid acyltransferase (LPAAT), diacylglycerol acyltransferase (DGAT), phospholipid diacylglycerol acyltransferase (PDAT), diacylglyceroldiacylglycerol transacylase (DDAT), and lysophospholipid acyltransferase (LPLAT). LPLATs can have activity as lysophosphophatidylethanolamine acyltransferase (LPEAT) and lysophosphatidylcholine acyltransferase (LPCAT).

The term “expression control sequence” as used herein refers to a nucleic acid sequence which is capable of governing, i.e., initiating and controlling, transcription of a nucleic acid sequence of interest, in the present case the nucleic sequences recited above. Such a sequence usually comprises or consists of a promoter or a combination of a promoter and enhancer sequences. Expression of a polynucleotide comprises transcription of the nucleic acid molecule, for example, into a translatable mRNA. Additional regulatory elements may include transcriptional as well as translational enhancers. The following promoters and expression control sequences may be, for example, used in an expression vector according to the present disclosure. The cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq, T7, T5, T3, gal, trc, ara, SP6, λ-PR or λ-PL promoters are, for example, used in Gram-negative bacteria. In some embodiments, for Gram-positive bacteria, promoters amy and SPO2 may be used. In some embodiments, yeast or fungal promoters ADC1, AOX1r, GAL1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH may be used. In some embodiments, for animal cell or organism expression, the promoters CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer may be used. From plants the promoters CaMV/35S (Franck 1980, Cell 21: 285-294], PRP1 (Ward 1993, Plant. Mol. Biol. 22), SSU, OCS, lib4, usp, STLS1, B33, nos or the ubiquitin or phaseolin promoter. In some embodiments, inducible promoters may be used, such as the promoters described in EP 0388186 A1 (i.e. a benzylsulfonamide-inducible promoter), Gatz 1992, Plant J. 2:397-404 (i.e. a tetracyclin-inducible promoter), EP 0335528 A1 (i.e. an abscisic-acid-inducible promoter) or WO 93/21334 (i.e., an ethanol- or cyclohexenol-inducible promoter). Further suitable plant promoters are the promoter of cytosolic FBPase or the ST-LSI promoter from potato (Stockhaus 1989, EMBO J. 8, 2445), the phosphoribosyl-pyrophosphate amidotransferase promoter from Glycine max (Genbank accession No. U87999) or the node-specific promoter described in EP 0249676 A1. In some embodiments, promoters which enable the expression in tissues which are involved in the biosynthesis of fatty acids are used. In some embodiments, seed-specific promoters are used, such as the USP promoter in accordance with the practice, but also other promoters such as the LeB4, DC3, phaseolin or napin promoters. In some embodiments, seed-specific promoters which can be used for monocotyledonous or dicotyledonous plants and which are described in U.S. Pat. No. 5,608,152 (napin promoter from oilseed rape), WO 98/45461 (oleosin promoter from Arobidopsis, U.S. Pat. No. 5,504,200 (phaseolin promoter from Phaseolus vulgaris), WO 91/13980 (Bce4 promoter from Brassica), by Baeumlein et al., Plant J., 2, 2, 1992:233-239 (LeB4 promoter from a legume), these promoters being suitable for dicots, may be used. The following promoters are suitable for monocots: lpt-2 or lpt-1 promoter from barley (WO 95/15389 and WO 95/23230), hordein promoter from barley and other promoters which are suitable, and which are described in WO 99/16890. In principle, it is possible to use all-natural promoters together with their regulatory sequences, such as those mentioned above, for the novel process. Likewise, it is possible and advantageous to use synthetic promoters, either additionally or alone, especially when they mediate a seed-specific expression, such as, for example, as described in WO 99/16890. In a particular embodiment, seed-specific promoters are utilized to enhance the production of the desired PUFA or VLC-PUFA.

The term “operatively linked” as used herein means that the expression control sequence and the nucleic acid of interest are linked so that the expression of the said nucleic acid of interest can be governed by the said expression control sequence, i.e. the expression control sequence shall be functionally linked to the said nucleic acid sequence to be expressed. Accordingly, the expression control sequence and, the nucleic acid sequence to be expressed may be physically linked to each other, e.g., by inserting the expression control sequence at the 5′end of the nucleic acid sequence to be expressed. Alternatively, the expression control sequence and the nucleic acid to be expressed may be merely in physical proximity so that the expression control sequence is capable of governing the expression of at least one nucleic acid sequence of interest. The expression control sequence and the nucleic acid to be expressed are, in some embodiments, separated by not more than 500 bp, 300 bp, 100 bp, 80 bp, 60 bp, 40 bp, 20 bp, 10 bp or 5 bp.

Polynucleotides of the present disclosure can include, in addition to a promotor, a terminator sequence operatively linked to polynucleotides which encode the enzymes, e.g., the desaturases and/or elongases, described herein.

The term “terminator” as used herein refers to a nucleic acid sequence which is capable of terminating transcription. These sequences will cause dissociation of the transcription machinery from the nucleic acid sequence to be transcribed. In some embodiments, the terminator shall be active in plants and, in particular, in plant seeds. Suitable terminators are known in the art and include polyadenylation signals such as the SV40-poly-A site or the tk-poly-A site or one of the plant specific signals indicated in Loke et al. (Loke 2005, Plant Physiol 138, pp. 1457-1468), downstream of the nucleic acid sequence to be expressed.

Recombinant nucleic acid molecules that encode desaturases and elongases described in FIG. 1 are suitable for use in embodiments of the present disclosure. As used herein, “recombinant” means the combination of nucleic acid sequences using techniques available to those of ordinary skill in molecular biology, to produce one or more expression cassette(s) (alternatively designated herein as gene constructs) or one or more vector(s) comprising polynucleotides encoding the desaturases and elongases described in FIG. 1, which are operably linked with expression control sequences such as promoters, to effect expression of the desaturase and elongase polynucleotides in a host cell.

Disclosed herein are recombinant polynucleotides (such as T-DNAs) for expression of desaturases and elongases in a Brassica plant. In some embodiments, a T-DNA comprises a left and a right border element and at least one expression cassette comprising a promotor, operatively linked to polynucleotides encoding various combinations of the desaturases and elongases, and downstream thereof other regulatory elements including but not limited to a terminator.

A “T-DNA” as used herein is a nucleic acid capable of eventual integration into the genetic material (genome) of a Brassica plant through transformation using methods available to those skilled in the art of molecular biology.

For example, a T-DNA suitable for use in embodiments of the present disclosure may be comprised in a circular nucleic acid, e.g. a plasmid, such that an additional nucleic acid section is present between the left and right border elements. The additional nucleic acid section may include one or more genetic elements for replication of the total nucleic acid, i.e. the nucleic acid molecule comprising the T-DNA and the additional nucleic acid section, in one or more host microorganisms, for example, in a microorganism of genus Escherichia, such as E. coli, and/or Agrobacterium. Such circular nucleic acids comprising a T-DNA of the present disclosure are particularly useful as transformation vectors.

In some embodiments, the T-DNA length is sufficiently large to introduce a number of enzymes, e.g. desaturase and elongase, genes in the form of expression cassettes, such that each individual gene is operably liked to at least one promotor and at least one terminator, as is shown in the examples below.

A T-DNA can comprise the coding sequences of one or more single genes. For example, T-DNA comprising the coding sequences of one or more single genes can be combined with other T-DNA comprising one or more other genes. The T-DNAs suitable for use in embodiments of the present disclosure may comprise one or more expression cassettes encoding for one or more d5Des, one or more d6Elo, one or more d5Des, one or more o3Des, one or more d5Elo and one or more d4Des, for example, for at least one CoA-dependent D4Des and one phospholipid-dependent d4Des. In some embodiments, the T-DNA encodes also one or more d12Des.

In one embodiment, the Brassica plant of the present disclosure or a part thereof (e.g., root, stem, leave, seed, flower, cell etc.) comprises one or more T-DNAs which encode for at least two d6Des, at least two d6Elo, and/or at least two o3Des. In one embodiment, the Brassica plant or a part thereof described herein includes a T-DNA comprising one or more expression cassettes encoding at least one CoA-dependent d4Des and at least one phospholipid dependent d4Des.

In one embodiment, at least one T-DNA suitable for use comprises an expression cassette which encodes at least one d12Des. In one embodiment, the T-DNA or T-DNAs comprise one or more expression cassettes encoding one or more one or more d5Des (e.g., delta 5 desaturase from Thraustochytrium sp., Tc_GA), o3Des (e.g., omega 3 desaturase from Pythium irregular, Pir_GA), d6Elo (delta 6 elongase from Thalassiosira pseudonana, Tp_GA) and/or d6Elo (e.g., delta-6 elongase from Physcomitrella patens, Pp_GA).

According to the disclosure, the T-DNA may also comprise, instead of one or more of the coding sequences discussed herein, a functional homolog thereof. A functional homolog of a coding sequence is a sequence coding for a polypeptide having the same metabolic function as the replaced coding sequence. As a non-limiting example, a functional homolog of a delta-5-desaturase would be another delta-5-desaturase, and a functional homolog of a delta-5-elongase would be another delta-5-elongase. A functional homolog of a plant seed specific promotor is another plant seed specific promotor. The functional homolog of a terminator, correspondingly, is a sequence for ending transcription of a nucleic acid sequence.

Certain T-DNA sequences suitable for use in embodiments of the present disclosure are described in PCT/EP2015/076632 (published as WO/2016/075327).

In some embodiments, constructs comprising a T-DNA vector comprising certain desaturases and elongases described herein can be transformed into a plant cell by microorganism-mediated transformation, for example, by Agrobacterium-mediated transformation. In some embodiments, the microorganism is a disarmed strain of genus Agrobacterium, such as species Agrobacterium tumefaciens or species Agrobacterium rhizogenes. Suitable Agrobacterium strains for use are for example described in WO06024509A2, and methods for plant transformation using such microorganisms are for example described in WO13014585A1, incorporated herein by reference.

The term “vector” encompasses phage, plasmid, viral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Moreover, the term also relates to targeting constructs which allow for random or site-directed integration of the targeting construct into genomic DNA. Such target constructs, in some embodiments, comprise DNA of sufficient length for either homolgous or heterologous recombination as described in detail below. The vector suitable for use in some embodiments further comprises selectable markers for propagation and/or selection in a host. The vector may be incorporated into a host cell by various techniques well known in the art. It is to be understood that the vector may further comprise nucleic acid sequences which allow for homologous recombination or heterologous insertion. Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection”, conjugation and transduction, as used in the present context, are intended to comprise a multiplicity of prior-art processes for introducing foreign nucleic acid (for example DNA) into a host cell, including calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, carbon-based clusters, chemically mediated transfer, electroporation or particle bombardment. Suitable methods for the transformation or transfection of host cells, including plant cells, can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and other laboratory manuals, such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, Ed.: Gartland and Davey, Humana Press, Totowa, N.J. Alternatively, a plasmid vector may be introduced by heat shock or electroporation techniques. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells.

In some embodiments, the vector referred to herein is suitable as a cloning vector, i.e. replicable in microbial systems. Such vectors ensure efficient cloning in bacteria and, in some embodiments, yeasts or fungi and make possible the stable transformation of plants. Those which must be mentioned are, in particular, various binary and co-integrated vector systems which are suitable for the T DNA-mediated transformation. Such vector systems are characterized in that they contain at least the vir genes, which are involved in the Agrobacterium-mediated transformation, and the sequences which delimit the T-DNA (T-DNA border). These vector systems, in some embodiments, also comprise further cis-regulatory regions such as promoters and terminators and/or selection markers with which suitable transformed host cells or organisms can be identified. While co-integrated vector systems have vir genes and T-DNA sequences arranged on the same vector, binary systems are based on at least two vectors, one of which bears vir genes, but no T-DNA, while a second one bears T-DNA, but no vir gene. As a consequence, the last-mentioned vectors are relatively small, easy to manipulate and can be replicated both in E. coli and in Agrobacterium. These binary vectors include vectors from the pBIB-HYG, pPZP, pBecks, pGreen series. In some embodiments, used in accordance with the disclosure are Bin19, pBI101, pBinAR, pGPTV and pCAMBIA. An overview of binary vectors and their use can be found in Hellens et al, Trends in Plant Science (2000) 5, 446-451. Furthermore, by using appropriate cloning vectors, the polynucleotides can be introduced into host cells or organisms such as plants or animals and, thus, be used in the transformation of plants, such as those which are published, and cited, in: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Fla.), chapter 6/7, pp. 71-119 (1993); F. F. White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus 1991, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42, 205-225. The binary BAC (BiBAC) vector, suitable for transforming large T-DNAs into plants, is described in U.S. Pat. Nos. 5,733,744 and 5,977,439.

An expression vector, i.e. a vector which comprises the polynucleotide of the disclosure having the nucleic acid sequence operatively linked to an expression control sequence (also called “expression cassette”) allowing expression in prokaryotic plant cells or isolated fractions thereof.

A Brassica plant or seed may comprise, integrated in its genome, a T-DNA capable of effecting expression of polynucleotides expressing the desaturases and elongases, such as the desaturases and elongases described in FIG. 1.

In some embodiments, the plants of the present disclosure are transgenic, i.e. they comprise genetic material not present in corresponding wild type plant or arranged differently in corresponding wild type plant, for example differing in the number of genetic elements. For example, the plants of the present disclosure can comprise promotors also found in wild type plants, but the plants of the present disclosure comprise such promotor operatively linked to a coding sequence such that this combination of promotor and coding sequence is not found in the corresponding wild type plant.

The Brassica plants of the present disclosure may comprise one or more T-DNA(s) described herein comprising expression cassettes which include one or more genes encoding for one or more d5Des, one or more d6Elo, one or more d5Des, one or more o3Des, one or more d5Elo and one or more D4Des, such as for at least one CoA-dependent D4Des and one phospholipid-dependent d4Des. In one embodiment, at least one T-DNA comprises an expression cassette which encodes for at least one d12Des. In one embodiment, the T-DNA or T-DNAs comprise one or more expression cassettes encoding one or more d5Des (e.g., delta 5 desaturase from Thraustochytrium sp., Tc_GA), o3Des (e.g., omega 3 desaturase from Pythium irregular, Pir_GA), d6Elo (delta 6 elongase from Thalassiosira pseudonana, Tp_GA) and/or d6Elo (e.g., delta-6 elongase from Physcomitrella patens, Pp_GA).

Seeds of an event described in the example below have been deposited at ATCC under the provisions of the Budapest treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, i.e. seeds of event “LBFLFK”=ATCC Designation “PTA-121703” (LBFLFK as described in PCT/EP2015/076632 (published as WO/2016/075327) and US 20180298400).

In some embodiments, a Brassica plant described herein can be produced using methods described in WO 2004/071467, WO 2015/089587 or WO 2016/075327, for producing Brassica lines. In some embodiments, a Brassica plant described herein can be produced using methods described in U.S. Pat. No. 7,807,849 B2 for producing Arabidopsis lines. In some embodiments, a Brassica plant described herein can be produced using methods described in WO 2013/153404, for producing Camelina lines.

In some embodiments, the Brassica plants provided herein can be a Brassica plant line. The term “line” refers to a group of plants that displays little to no genetic variation for at least one trait among individuals sharing that designation.

The Brassica plants and seeds disclosed herein are, in some embodiments, of a species comprising a genome of one or two members of the species Brassica oleracea, Brassica nigra, and Brassica rapa. In some embodiments, the Brassica plants and seeds disclosed herein are of the species Brassica napus, Brassica carinata, Brassica juncea, Brassica oleracea, Brassica nigra, or Brassica rapa. In some embodiments, the plants and seeds are of the species Brassica napus and Brassica carinata.

In some embodiments, a plant provided herein is a plant found in the “Triangle of U”, i.e. a plant of genus Brassica: Brassica napus (AA CC genome; n=19), which is an amphidiploid plant of the Brassica genus, but is thought to have resulted from hybridization of Brassica rapa (AA genome; n=10) and Brassica oleracea (CC genome; n=9). Brassica juncea (AA BB genome; n=18) is an amphidiploid plant of the Brassica genus that is generally thought to have resulted from the hybridization of Brassica rapa and Brassica nigra (BB genome; n=8). Under some growing conditions, B. juncea may have certain superior traits to B. napus. These superior traits may include higher yield, better drought and heat tolerance and better disease resistance. Brassica carinata (BB CC genome; n=17) is an amphidiploid plant of the Brassica genus but is thought to have resulted from hybridization ofBrassica nigra and Brassica oleracea. Under some growing conditions, B. carinata may have superior traits to B. napus.

In some embodiments, the Brassica plant provided herein is a “canola” plant. Canola herein generally refers to plants of Brassica species that have less than 2% (e.g., less than 11%, 0.5%, 0.2% or 0.1%) erucic acid (delta 13-22:1) by weight in seed oil and less than about 30 micromoles (e.g., less than 30, 25, 20 15, or 10 micromoles) of glucosinolates per gram of oil free meal (meal fraction). Typically, canola oil may include saturated fatty acids known as palmitic acid and stearic acid, a monounsaturated fatty acid known as oleic acid, and polyunsaturated fatty acids known as linoleic acid and linolenic acid. Canola oil may contain less than about 7% (w/w) total saturated fatty acids (mostly palmitic acid and stearic acid) and greater than 40% (w/w) oleic acid (as percentages of total fatty acids). Traditionally, canola crops include varieties of Brassica napus and Brassica rapa. Non-limiting exemplary Brassica plants of the present disclosure are spring canola (Brassica napus subsp. oleifera var. annua) and winter canola (Brassica napus subsp. oleifera var. biennis). Furthermore, a canola quality Brassica juncea variety, which has oil and meal qualities similar to other canola types, has been added to the canola crop family (U.S. Pat. Nos. 6,303,849; 7,423,198; all of which are incorporated herein by reference). Likewise, it is possible to establish canola quality B. carinata varieties by crossing canola quality variants of Brassica napus with Brassica nigra and appropriately selecting progeny thereof, optionally after further back-crossing with B. carinata, B. napus, and/or B. nigra.

This method allows to effectively incorporate genetic material of other members of family Brassicaceae, such as genus Brassica, into the genome of a plant comprising a T-DNA disclosed herein. The method is particularly useful for combining an event comprising a T-DNA with genetic material responsible for beneficial traits exhibited in other members of family Brassicaceae. Beneficial traits of other members of family Brassicaceae are exemplarily described herein, other beneficial traits or genes and/or regulatory elements involved in the manifestation of a beneficial trait may be described elsewhere. In some embodiments, a Brassica plant that produces higher levels of linolenic acid can be crossed with a Brassica plant that produces one or more of EPA, DPA, and DHA, such that the progeny produces higher levels of one or more of EPA, DPA and/or DHA than either parent plant. In some embodiments, a Brassica plant that produces low levels of linolenic acid can be crossed with a Brassica plant that produces one or more of EPA, DPA and/or DHA, and surprisingly, the progeny can produce higher levels of one or more of EPA, DPA and/or DHA than either parent plant. In some embodiments, a Brassica plant that produces higher levels of linoleic acid can be crossed with a Brassica plant that produces one or more of EPA, DPA and/or DHA, such that the progeny produces higher levels of one or more of EPA, DPA and/or DHA than either parent plant. In some embodiments, a Brassica plant that produces low levels of linoleic acid can be crossed with a Brassica plant that produces one or more of EPA, DPA and/or DHA, and surprisingly, the progeny can produce higher levels of one or more of EPA, DPA and/or DHA than either parent plant. In some embodiments, a Brassica plant that produces mid-range levels of linoleic acid can be crossed with a Brassica plant that produces one or more of EPA, DPA and/or DHA, and surprisingly, the progeny can produce higher levels of one or more of EPA, DPA and/or DHA than either parent plant, and in some embodiments, higher levels of DHA and/or EPA than a plant resulting from a cross of a high linoleic acid producing Brassica parent with a Brassica plant that produces one or more of EPA, DPA and/or DHA.

In some embodiments, the parent plant not comprising the T-DNA described herein is a parent that produces high linolenic acid, such as the rrm1367-003 line described in WO2015/066082. In some embodiments, the parent plant not comprising the T-DNA described herein can be a parent that produces low linolenic acid. In some embodiments, the parent plant not comprising the T-DNA described herein is a parent that produces low linoleic acid.

In some embodiments, a parent plant can have all or part of at least one genomic sequence of a B. napus parent genome that confers higher PUFA content, where the genomic sequence is selected from the group consisting of: a) the genomic sequence on chromosome N1 between nucleotide positions 8879780 and 11922690; b) the genomic sequence on chromosome N1 between nucleotide positions 22823086 and 24045492; and c) the genomic sequence on chromosome N6 between nucleotide positions 19156645 and 20846412. In the present disclosure, nucleotide positions within a given chromosome are based on the position in the genomic sequence of Brassica napus cultivar DH12075.

In some embodiments, a Brassica plant produced as described herein comprises (i) a T-DNA as described herein and (ii) all or part of at least one genomic sequence of a B. napus parent genome that confers higher PUFA content, where the genomic sequence is selected from the group consisting of: a) the genomic sequence on chromosome N1 between nucleotide positions 8879780 and 11922690; b) the genomic sequence on chromosome N1 between nucleotide positions 22823086 and 24045492; and c) the genomic sequence on chromosome N6 between nucleotide positions 19156645 and 20846412.

In some embodiments, the genomic sequence of a B. napus parent genome that confers higher PUFA can include, for example, from 25 to 50, 25 to 100, 50 to 200, 100 to 500, 250 to 1,000, 500 to 5,000, 2,000 to 10,000, 5,000 to 20,000, 10,000 to 100,000, 50,000 to 400,000, 25,000 to 1,000,000, 100,000 to 1,000,000, 200,000 to 1,000,000, or 500 to 1,000,000 contiguous nucleotides or longer of a region of chromosome N1 (e.g., the genomic sequence on chromosome N1 between nucleotide positions 8879780 and 11922690 and/or the genomic sequence on chromosome N1 between nucleotide positions 22823086 and 24045492) and/or a region of chromosome N6 (e.g., the genomic sequence on chromosome N6 between nucleotide positions 19156645 and 20846412).

In some embodiments, one or more single nucleotide polymorphisms (SNPs) can be present in all or part of at least one genomic sequence of a B. napus parent genome that confers higher PUFA content. The presence of one or more such SNPs can be used in selecting suitable parents and progeny. A SNP can occur within coding and non-coding regions, including exons, introns, and untranslated sequences. Examples of SNPs include substitutions of one or more nucleotides, deletions of one or more nucleotides, and insertions of one or more nucleotides. In some embodiments, a nucleotide substitution can be a transition, in which a purine nucleotide is substituted for another purine (e.g., A to G or G to A), or a pyrimidine nucleotide is substituted for another pyrimidine (e.g., C to T or T to C). In some embodiments, a nucleotide substitution can be a transversion, in which a purine nucleotide is substituted for a pyrimidine or a pyrimidine nucleotide is substituted for a purine nucleotide (e.g., G to T, or C to G). A nucleotide substitution within a coding sequence that results in the substitution of an amino acid also can be referred to as a non-synonymous SNP.

In some embodiments, a Brassica plant can include all or part of the genomic sequence on chromosome N1 between nucleotide positions 8879780 and 11922690 that confers higher PUFA content. In some embodiments, the genomic sequence that confers higher PUFA content can include one or more SNPs (e.g., two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more different SNPs) between nucleotide positions 8879780 and 11922690 on chromosome N1. Table 9 provides examples of SNPs within chromosome N1 that are distributed throughout the genomic sequence between nucleotide positions 8879780 and 11922690, including SNPs at positions 8,952,616, 9,040,901, 9,046,609, 9,048,617, 9,136,686, 9,143,608, 9,248,592, 9,347,120, 9,352,326, 9,454,361, 9,549,523, 9,641,936, 9,652,028, 9,794,198, 9,847,417, 9,921,975, 9,952,792, 10,052,015, 10,402,684, 10,425,211, 10,558,464, 10,613,015, 10,659,284, 10,706,805, 10,748,492, 10,852,010, 11,007,740, 11,047,958, 11,150,929, 11,269,217, 11,343,118, 11,455,979, 11,565,970, 11,659,776, 11,726,807, and 11,850,103. Table 10 provides examples of SNPs in candidate genes (e.g., genes that encode products involved in lipid biosynthesis or a related pathway in the parent which increases PUFA) within chromosome N1 between nucleotide positions 8879780 and 11922690 including positions 9,136,686, 9,641,936, 10,613,015, 9,040,901, 9,048,617, 9,352,326, 9,921,975, and 10,706,805. In some embodiments, all or part of the genomic sequence on chromosome N1 between nucleotide positions 8879780 and 11922690 that confers higher PUFA content can include one or more non-synonymous SNPs at positions 9,136,686, 9,143,608, 9,454,361, 9,952,792, 9,549,523 9,641,936, 9,652,028, 10,613,015, 9,352,326, 9,794,198, 9,847,417, 9,921,975, 10,402,684, 10,706,805, 10,659,284, 10,748,492, 11,007,740, 11,047,958, 11,150,929, 11,269,217, 11,455,979, 11,659,776, or 11,850,103.

In some embodiments, a Brassica plant can include all or part of the genomic sequence on chromosome N1 between nucleotide positions 22,823,086 and 24,045,492 that confers higher PUFA content. The genomic sequence associated with higher PUFA content can include one or more SNPs (e.g., two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more different SNPs) between nucleotide positions nucleotide positions 22,823,086 and 24,045,492 on chromosome N1. Table 11 provides examples of SNPs within chromosome N1 that are distributed throughout the genomic sequence between nucleotide positions 22,823,086 and 24,045,492, including SNPs at positions 22,823,086, 22,880,595, 22,902,670, 22,949,738, 23,011,207, 23,044,228, 23,099,592, 23,176,771, 23,201,595, 23,257,618, 23,302,268, 23,367,822, 23,380,089, 23,457,696, 23,520,607, 23,552,773, 23,598,941, 23,670,623, 23,682,848, 23,745,365, 23,792,572, 23,855,829, 23,910,029, 23,947,522, and 24,021,883. Table 12 provides examples of SNPs in candidate genes (e.g., genes that encode products involved in lipid biosynthesis or a related pathway in the parent which increases PUFA) within chromosome N1 between nucleotide positions 22,823,086 and 24,045,492 including positions 23,089,542, 23,089,635, 23,090,743, 23,090,785, 23,091,367, 23,092,042, 23,150,402, 23,150,595, 23,155,220, 23,155,766, 23,314,197, 23,318,357, 23,343,089, 23,679,276, 23,679,287, 23,679,396, 23,886,929, 23,925,895, 23,963,309, 24,029,270, 24,029,279, and 24,029,294. In some embodiments, all or part of the genomic sequence on chromosome N1 between nucleotide positions 22,823,086 and 24,045,492 that confers higher PUFA content can include one or more non-synonymous SNPs at positions 23,089,542, 23,089,635, 23,090,743, 23,090,785, 23,091,367, 23,092,042, 23,099,592, 23,150,402, 23,150,595, 23,155,220, 23,155,766, 23,201,595, 23,257,618, 23,314,197, 23,318,357, 23,380,089, 23,457,696, 23,520,607, 23,552,773, 23,598,941, 23,679,276, 23,679,287, 23,679,396, 23,682,848, 23,745,365, 23,855,829, 23,925,895, 23,947,522, 24,021,883, 24,029,270, 24,029,279, or 24,029,294.

In some embodiments, a Brassica plant can include all or part of the genomic sequence on chromosome N6 between nucleotide positions 19,156,645 and 20,846,412 that confers higher PUFA content. The genomic sequence that confers higher PUFA content can include one or more SNPs (e.g., two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more different SNPs) between nucleotide positions nucleotide positions 19,156,645 and 20,846,412 on chromosome N6. Table 13 provides examples of SNPs within chromosome N6 that are distributed throughout the genomic sequence between nucleotide positions 19,156,645 and 20,846,412, including SNPs at positions 19,156,645, 19,199,109, 19,325,186, 19,402,086, 19,513,420, 19,583,431, 19,601,021, 19,706,563, 19,800,643, 19,906,666, 20,000,119, 20,095,002, 20,205,211, 20,300,571, 20,406,148, 20,407,023, 20,505,840, 20,601,198, 20,631,917, and 20,702,631. Table 14 provides examples of SNPs in candidate genes (e.g., genes that encode products involved in lipid biosynthesis or a related pathway in the parent which increases PUFA) within chromosome N6 between nucleotide positions 19,156,645 and 20,846,412 including positions 19,336,744, 19,336,819, 19,337,615, 19,350,156, 19,353,584, 19,353,648, 19,353,749, 19,476,836, 19,783,834, 19,784,007, 19,784,367, 19,784,633, 19,784,672, 19,784,688, 19,784,733, 19,800,525, 20,191,826, 20,300,548, 20,375,643, 20,766,637, 20,769,461, 20,770,769, 20,823,998, 20,825,959, 20,826,301, 20,827,570, 20,827,573, and 20,912,356. In some embodiments, all or part of the genomic sequence on chromosome N6 between nucleotide positions 19,156,645 and 20,846,412 that confers higher PUFA content can include one or more non-synonymous SNPs at positions 19,325,186, 19,336,744, 19,336,819, 19,337,615, 19,350,156, 19,353,584, 19,353,648, 19,353,749, 19,402,086, 19,513,420, 19,783,834, 19,784,007, 19,784,367, 19,784,633, 19,784,672, 19,784,688, 19,784,733, 19,800,525, 19,906,666, 20,000,119, 20,095,002, 20,300,548, 20,375,643, 20,766,637, 20,769,461, 20,770,769, 20,823,998, 20,825,959, 20,826,301, 20,827,570, or 20,827,573.

In some embodiments, a Brassica plant can include all or part of the genomic sequence on chromosome N1 between nucleotide positions 8879780 and 11922690 that confers higher PUFA content and all or part of the genomic sequence on chromosome N1 between nucleotide positions 22,823,086 and 24,045,492 that confers higher PUFA content. Examples of SNPs that can be found in each of these regions are described above.

In some embodiments, a Brassica plant can include all or part of the genomic sequence on chromosome N1 between nucleotide positions 8879780 and 11922690 that confers higher PUFA content and all or part of the genomic sequence on chromosome N6 between nucleotide positions 19,156,645 and 20,846,412 that confers higher PUFA content. Examples of SNPs that can be found in each of these regions are described above.

In some embodiments, a Brassica plant can include all or part of the genomic sequence on chromosome N1 between nucleotide positions 22,823,086 and 24,045,492 that confers higher PUFA content and can include all or part of the genomic sequence on chromosome N6 between nucleotide positions 19,156,645 and 20,846,412 that confers higher PUFA content. Examples of SNPs that can be found in each of these regions are described above.

In some embodiments, the Brassica plants provided herein (e.g., Brassica napus plants) produce seeds with an EPA content of at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 15%, at least about 16%, or at least about 17% based on total weight of fatty acids (C14-C22). In some embodiments, the EPA content can range from at least about 6% to about 18% (e.g., about 8% to about 18%, about 10% to about 18%, about 12% to about 18%, about 12.5% to about 17.5%, or about 12.5% to about 15%).

In some embodiments, the Brassica plants provided herein (e.g., Brassica napus plants) produce seeds with a DHA content of at least about 0.9%, at least about 1.0%, at least about 1.2%, at least about 1.3%, at least about 1.4%, at least about 1.5%, at least about 1.6%, at least about 1.7%, at least about 1.8%, at least about 1.9%, or at least about 2% based on total weight of fatty acids (C14-C22). In some embodiments, the DHA content can range from about 0.9% to about 2% (e.g., about 0.9% to about 1.5%, about 1.0% to about 2.0%, about 1.0% to about 1.9%, or about 1.2% to about 1.9%).

In some embodiments, the Brassica plants provided herein (e.g., Brassica napus plants) produce seeds with a DPA content of at least about 3.5%, at least about 4.0%, at least about 4.5%, at least about 5.0%, at least about 5.5%, or at least about 6% based on total weight of fatty acids (C14-C22). In some embodiments, the DPA content can range from about 3.5% to about 6%, or from about 4.0% to about 6% (e.g., about 4% to about 5%, about 4.75% to about 6.0%, about 4.75% to about 5.75%, or about 5.0% to about 6.0%).

In some embodiments, the Brassica plants provided herein (e.g., Brassica napus plants) produce seeds with a DHA content of about 0.5% to about 2.8% and/or an EPA content of about 3.5% to about 15.0% EPA. In some embodiments, the DHA content can range from about 0.9 to about 1.5% and/or an EPA content of about 12.5% to about 15.0%.

In some embodiments, the Brassica plants provided herein (e.g., Brassica napus plants) produce seeds with an EPA, DPA, and DHA content of at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, or at least about 23%. In some embodiments, the Brassica plants produce seeds with an EPA, DPA, and DHA content that ranges from about 19% to about 24% (e.g., about 20% to about 24%, about 20% to about 23%, or about 21% to about 23%).

Brassica plants described herein that produce seeds having higher levels of EPA, DPA, and/or DHA compared to corresponding control plants (e.g., plants lacking the T-DNA expression construct and/or lacking the genomic sequence(s) from chromosome N1 and/or chromosome N6 that confers higher PUFA content), and the parts thereof, can be used for feed purposes such as aquaculture feed, e.g. as described in AU2011289381A and members of the patent family thereof.

In some embodiments, a Brassica plant provided herein is tolerant of an herbicide such as an imidazolinone, dicamba, cyclohexanedione, a sulfonylurea, glyphosate, glufosinate, phenoxy propionic acid, L-phosphinothricin, a triazolinone, a triazolpyrimidine, a pyrimidinylthiobenzoate, and benzonitrile. For example, Brassica plants can include a polynucleotide that encodes a product (e.g., a mutant acetohydroxyacid synthase) that confers resistance to an herbicide (e.g., an imidazolinones, a sulfonylureas, a pyrimidinylthiobenzoate, a triazolinone, or a triazolopyrimidine). See, for example, Tans et al., Pest Manag Sci. 61(3):246-57 (2005) and Hu et al., PLoS One. 12(9): e0184917 (2017).

The present disclosure also relates to oil comprising a polyunsaturated fatty acid obtainable from the plants described herein. The term “oil” refers to a fatty acid mixture comprising unsaturated and/or saturated fatty acids which are esterified to triglycerides. In some embodiments, the triglycerides in the oil of the disclosure comprise PUFA or VLC-PUFA moieties as referred to above. The amount of esterified PUFA and/or VLC-PUFA is, in some embodiments, approximately 30%, or at least 50%, or at least 60%, 70%, 80% or more. The oil may further comprise free fatty acids, such as the PUFA and VLC-PUFA referred to above.

The oils according to the disclosure can have an EPA content of at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 15%, at least about 16%, or at least about 17% based on the total fatty acid content. The oils according to the disclosure can have a DHA content of at least about 0.9%, at least about 1.0%, at least about 1.2%, at least about 1.3%, at least about 1.4%, at least about 1.5%, at least about 1.6%, at least about 1.7%, at least about 1.8%, at least about 1.9%, or at least about 2% based on the total fatty acid content. In some embodiments, the oils of the disclosure can have a DHA content of about 0.5 to about 2.8% and/or EPA content of about 3.5% to about 15.0% EPA. In some embodiments, an oil of the disclosure can have a DHA content of about 0.9 to about 1.5% and/or an EPA content of about 12.5% to about 15.0% EPA. In some embodiments, an oil of the disclosure can have an EPA, DPA, and DHA content of at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, or at least about 23%. In some embodiments, an oil of the disclosure can have an EPA, DPA, and DHA content that ranges from about 19% to about 24% (e.g., about 20% to about 24%, about 20% to about 23%, or about 21% to about 23%).

In some embodiments, the plants, such as the progeny, can be hybrids or inbreds. The term hybrid relates to a cultivar or plant-breeding progeny based upon the controlled cross-pollination between or among distinct parent lines, so that the resulting seed inherits its genetic composition from those parent lines. Seed for a particular hybrid can be repeatedly and predictably produced when repeatedly making controlled cross-pollinations from the same stable female and male parent genotypes. While inbred refers to a relatively stable plant genotype resulting from doubled haploids, successive generations of controlled self-pollination, successive generations of controlled backcrossing to a recurrent parent, or other method to develop homozygosity. Backcrossing refers to a process in which a breeder repeatedly crosses hybrid progeny back to one of the parents; for example, a first-generation hybrid F1 crossed back to one of the parental genotypes of the F1 hybrid. The production of hybrid plants is well known/available to an art worker.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES
Example 1: Genome Mapping to Identify Quantitative Trait Loci (QTL) that Increase EPA, DPA and/or DHA
Materials and Methods
Plant Propagation

Brassica plants were grown in a growth chamber (Conviron GR192) in Berger B7 soil with 100 ppm fertilizer at every watering (Jack's 20-20-20) from rosette stage through end of flowering. Chamber conditions were 16-hour day length with 22° C. day temperature and 19° C. night temperature, or at 28° C. day temperature and 15° C. night temperature. Seed was harvested at full maturity.

Cross Pollination

Homozygous PUFA donor LBFLFK (LBFLFK bears two insertions of the VC-LTM593-lqcz plasmid at two different loci; LBFLFK and the genetic elements of VC-LTM593-lqcz rc and the function of each element are provided in PCT/EP2015/076632 (published as WO/2016/075327) was used as pollen donor in a cross with many unique Brassica accessions to make F1 seed. The F1 seed fatty acid profile was determined by gas chromatography.

[Image Omitted]

Genotypic Analysis

Leaf samples were taken from each accession prior to flowering. DNA was extracted from each leaf sample using DNeasy minipreps (Qiagen) and was analyzed for single nucleotide polymorphisms (SNPs) by Illumina Infinium 60k Brassica array and KASP (competitive allele-specific polymerase chain reaction (PCR), LGC).

Phenotypic Analysis

Fatty acid profile of seed was measured by gas chromatography using ˜30 seeds and standard fatty acid methyl ester preparation (adapted from AOCS method Ce 1-62) immediately following crushing. GLC-566 (NuChek Prep) was used for the standard and fatty acid profiles were determined by ChemStation software (Agilent) as a percent of total fatty acids.

The fatty acid composition of seeds was determined by a modification of American Oil Chemist's Society (AOCS) protocol Ce 1-62. In the procedure fatty acids present as acylglycerols are converted to fatty acid methyl esters, which are analyzed by gas liquid chromatography (GLC or GC). For each sample to be analyzed 20-30 seeds are placed in a 15 ml centrifuge tube along with two steel ball bearings. The tube is capped and shaken for 30 seconds or until the seeds are visibly crushed. Approximately 0.6 mL of 2 N KOH in methanol is added to the tube, and the tube is shaken again for approximately one minute. The tube and its contents are placed in a water bath at 70+5° C. for 2 min. After removing the tube from the bath 4 mL of water saturated with sodium chloride and 2.0 mL of isooctane with 100 ppm of BHT are added, the tube is shaken and centrifuged for 1 min. in a tabletop centrifuge. A portion of the isooctane supernatant is transferred to a gas chromatographic (GC) vial and capped. Vials are stored at 0-4° C. until analysis, but no more than five days.

Fatty acid methyl esters were subject to analysis on a GC on an instrument equipped with a 20 m×0.18 mm×0.2 μm DB-225 (50% Cyanopropylphenyl) column from Agilent Technologies. An injector temperature of 250° C. was applied and 1 μl was injected with a split of 50:1 using 0.8 ml/min Hydrogen column flow (constant flow mode). Initial temperature is 190° C./0 min->15 C°/min->220° C.->220° C./9 min. and a flame ionization detector. The instrument is calibrated with a fatty acid methyl ester standard, such as NuChek Prep Catalog number GLC 566.

The content of fatty acids having from 14 carbon atoms (C14 fatty acids) to 24 carbon atoms (C24 fatty acids) is determined using the integrated peak area for each type of fatty acid reported normalized to the total peak area for those fatty acids.

The levels of particular acids are provided herein in percentages. Unless specifically noted otherwise, such percentages are weight percentages based on the total fatty acids in the seed oil, as calculated experimentally. Thus, for example, if a percentage of a specific species of fatty acid is provided, e.g., oleic acid, this is a w/w percentage based on the total fatty acids detected in the seed oil.

Genetic Mapping
QTL (Quantitative Trait Loci) Mapping

A high-density single nucleotide polymorphism (SNP) Illumina Infinium array containing 52,157 markers (Clarke et al. 2016) was used in this study to genotype a total of 288 Brassica accessions. DNA was isolated, quantified and hybridized to the array as described in the manufacturer's protocol (Illumina Inc., San Diego, Calif.). The arrays were scanned using an Illumina HiScan or BeadArray Reader, and SNP data were analyzed using the Genotyping module of the GenomeStudio software package. A total of 47,304 SNPs passed quality analysis and were used to perfonm the GWAS (Genome-Wide Association Study) analysis in the R environment (R Development Core Team, 2015). The GAPIT package, a genomic association and prediction integrated tool (Version 2; Lipka et al. 2012) and the GWAS function in the rrBLUP package (Endelman, 2011) were both used to identify genomic blocks conferring the PUFA phenotype including EPA, DPA and DHA content. Oil trait distribution and correlation analyses were also carried out in an R environment.

QTL Validation

Two backcrossing (BC₁) populations were developed from crosses between the PUFA donor line, Kumily LBFLFK, and two Brassica accessions carrying favorable alleles for EPA and DHA based on the association mapping results. Selections were genotyped to confirm copy number of the PUFA events. LBFLFK contains two PUFA loci (e.g., two insertions of the construct carrying the PUFA pathway). Lines that were homozygous for both loci or heterozygous for both loci were chosen for mapping. A total of 658 BC₁lines were selected from both populations (Table 1).

[Image Omitted]

TABLE 1

Number of homozygous and heterozygous lines selected

from each BC1 population and used for QTL mapping.

Line
# Selected

2
127 homozygous 2 loci, 130 heterozygous 2 loci

4
174 homozygous 2 loci, 227 heterozygous 2 loci

DNA from these 658 BC1 lines was genotyped with 1434 genome-wide Ion AmpliSeq sequencing (Life Technologies) SNP markers along with 85 KASP (LGC, UK) SNP markers located within the QTL regions identified from GWAS analysis. A subset of SNPs was polymorphic for each line.

Correlations between fatty acid composition and single SNP markers were calculated in Microsoft Excel (2010). Linkage maps of the experimental populations were constructed using the Kosambi function of JoinMap 3.0 (Kyazma). Quantitative trait loci (QTL) mapping was done in the R/qtl program of the R statistical package (Broman et al., 2003; Broman and Sen, 2009).

Results

Genome-Wide Association Study Mapping

FIG. 2 shows the distribution of EPA, DPA and DHA from 279 Brassica accessions heterozygous for each LBFLFK insertion (arrow shows the average content from the PUFA donor line, Kumily LBFLFK). Genome wide association study analyses identified two significant associations on the A01 chromosome (FIGS. 3, 4 and 5) and one significant association on A06 (FIG. 6). Specific SNP markers for the QTL are specified in Tables 3, 4, and 5. Haplotype analysis identified haplotypes correlated with PUFA traits, including two favorable haplotypes corresponding to the two QTLs on A01, respectively, which increase the content of EPA, DPA and DHA. For example, accessions carrying the favorable haplotype corresponding to QTL1 had an average 55% increase in EPA (6.70% compared to 4.32%), 101% increase in DPA (4.16% compared to 2.07%), and 121% increase in DHA (1.17% compared to 0.53%). Lines carrying the favorable haplotype for the QTL on A05 had an average 71% increase in EPA (7.28% compared to 4.26%), 54% increase in DPA (3.10% compared to 2.01%), and 163% increase in DHA (1.34% compared to 0.51%). And finally, lines carrying the favorable haplotype for the QTL on A06 had an average 53% increase in EPA (6.79% compared to 4.43%), 79% increase in DPA (3.70% compared to 2.07%), and 162% increase in DHA (1.44% compared to 0.55%).

QTL Validation

QTL scans of the BC1 population confirmed the presence of the loci identified in the GWAS study (QTL1, 2, and 3; Table 6). Here it can be seen that individuals heterozygous at each of the specified loci contained higher PUFA contents than individuals homozygous for the PUFA donor line, Kumily LBFLFK.

TABLE 2

Summary of EPA + DHA values of Brassica napus

lines carrying various combinations of QTL on A01.

Line
QTL1-A01
QTL2-A01
EPA + DHA

Brassica rapa 1*
+
+
14.7

Brassica napus 2
+
−
12.1

Brassica napus 3
+
+
11.6

Brassica napus 4
+
+
10.8

Brassica napus 5
+
+
10.7

Brassica napus 6
−
+
10.4

Brassica napus 7
+
−
9.8

Brassica napus 8
−
−
9.4

Brassica napus 9*
+
−
9.3

Brassica napus 10
+
−
9

“+” indicates the presence of the favorable (PUFA increasing) genotype and “−” indicates the alternative allele.

TABLE 3

SNP locations of the first genomic block (QTL1) on N01

SNP markers
Chromosome
Position in DH12075 (V3.0)

Bn-A01-p22949106
1
22452876

Bn-A01-p23195492
1
22672365

Bn-A01-p23253678
1
22730061

Bn-A01-p23259034
1
22735485

Bn-A01-p23614494
1
23132864

TABLE 4

SNP locations of the 2^ndgenomic block (QTL2) on N01

SNP markers
Chromosome
Position in DH12075 (V3.0)

Bn-A01-p7961620
1
8008195

Bn-A01-p7973418
1
8025818

Bn-A01-p7974551
1
8027772

Bn-A01-p7979458
1
8033912

Bn-A01-p7983241
1
8037687

Bn-A01-p7987687
1
8044316

TABLE 5

SNP location of the 3^rdgenomic block (QTL3) on N06

SNP markers
Chromosome
Position in DH12075 (3.0)

Bn-A06-p8634648
6
8317333

Bn-A06-p8697187
6
8381641

Bn-A06-p8697193
6
8381647

Bn-A06-p8732612
6
8395518

Bn-A06-p8766088
6
8429433

Bn-A06-p14664213
6
16607904

Bn-A06-p14809503
6
16742528

Bn-A06-p14813811
6
16746780

TABLE 6

Markers and average PUFA values for alternate genotypes at each locus.

Position in

SNP Marker
DH12075 (v3.0)
Genotype
C20:5
C22:5
C22:6
EPA + DPA + DHA

QTL1
Bn-A01-p14943499
14441849
CC
6.092
2.852
0.531
9.475

CT
6.364
2.986
0.581
9.932

Bn-A01-p21393260
21381690
GG
5.955
2.815
0.517
9.287

GA
7.106
3.242
0.639
10.988

23_199_N1_G277T
23302241
GG
5.993
2.835
0.519
9.347

TG
6.533
3.027
0.602
10.162

Bn-A01-p26447438
24568625
TT
6.008
2.853
0.516
9.378

GT
6.479
2.984
0.596
10.059

QTL2
Bn-A01-p7479483
7515195
CC
6.049
2.818
0.55
9.417

CA
6.374
2.987
0.561
9.922

Bn-A01-p9614730
9123417
GG
6.052
2.833
0.535
9.421

GA
6.411
2.992
0.577
9.98

Bn-A01-p9961553
9496833
CC
6.028
2.825
0.533
9.386

CT
6.4
2.986
0.576
9.962

Bn-A01-p14943499
14441849
CC
6.092
2.852
0.531
9.475

CT
6.364
2.986
0.581
9.932

QTL3
Bn-A06-p7499751
7197412
TT
5.548
2.608
0.494
8.649

GT
6.208
2.821
0.565
9.594

Bn-A06-p7670696
7381938
AA
5.501
2.591
0.493
8.585

GA
6.218
2.821
0.562
9.6

Bn-A06-p19185585
14926457
TT
5.573
2.607
0.499
8.679

CT
6.146
2.803
0.555
9.504

Bn-A06-p19333055
15086022
GG
5.539
2.59
0.499
8.628

AG
6.128
2.801
0.551
9.479

Bn-A06-p19562187
15302019
TT
5.568
2.61
0.498
8.676

GT
6.145
2.8
0.556
9.501

Bn-A06-p14813811
16746780
GG
5.597
2.614
0.502
8.713

TG
6.204
2.825
0.563
9.592

Bn-A06-p15712062
17641645
GG
5.524
2.571
0.499
8.594

TG
6.174
2.826
0.557
9.558

Bn-A06-p16015727
17961288
GG
5.566
2.609
0.49
8.664

AG
6.384
2.947
0.578
9.908

Bn-A06-p17114458
18994122
GG
5.528
2.585
0.49
8.602

AG
6.187
2.823
0.565
9.574

Bn-A06-p18500596
20405390
AA
5.52
2.613
0.48
8.613

GA
6.237
2.813
0.576
9.626

Bn-A06-p21490506
20769890
TT
5.565
2.626
0.479
8.669

CT
6.197
2.801
0.578
9.576

Subsequent fine mapping analysis was performed to validate and narrow the QTL regions for QTL1, 2 and 3 to the genomic regions that are identified in Table 7. Fine mapping was performed by crossing the Parent 1 non-PUFA/Parent 2 PUFA F₁with the elite female parent, backcrossing twice to elite female parent containing LBFLFK, selfing and using the BC₂S₂and BC₂S₃selfed populations to map. In each of these generations, selections were made using correlation of SNP genotype with VLC-PUFA as described in QTL Mapping.

Simultaneously, the QTL markers identified in mapping and fine mapping as described herein were used to introgress the QTL into elite parent lines, including the elite female parent shown in Table 8. For introgression, the elite parent line homozygous for LBFLFK (“Control” in Table 8) was crossed to Parent 2 which contained both LBFLFK and QTL1 and QTL2 and crossed to Parent 3 which contained both LBFLFK and QTL3. The resultant progeny were selected for the respective QTL and backcrossed twice to the elite parent line. The resultant progeny from each cross were then crossed to each other to combine LBFLFK, QTL1, QTL2 and QTL3 in a single plant. The progeny were selfed and plants homozygous for LBFLFK, QTL1, QTL2 and QTL3 were selected, selfed and were grown in chambers (28° C. day temperature and 15° C. night temperature), and the fatty acid profile was assessed in seeds harvested from the plants. As shown in Table 8, plants with QTL3 had an average 15% increase in overall PUFA content (18.07% compared to 15.67%), with an average 12% increase in EPA (12.66% compared to 11.32%), average 19% increase in DPA (4.26% compared to 3.57%), and average 46% increase in DHA (1.15% compared to 0.79%). Plants with both QTL1 and QTL2 had an average 10% increase in overall PUFA content (17.3% compared to 15.67%), with an average 12% increase in EPA (12.73% compared to 11.32%), average 2% increase in DPA (3.64% compared to 3.57%), and average 19% increase in DHA (0.94% compared to 0.79%). Plants with QTL1, QTL2, and QTL3 had an average 28% increase in overall PUFA content (20.04% compared to 15.67%), with an average 27% increase in EPA (14.32% compared to 11.32%), average 24% increase in DPA (4.41% compared to 3.57%), and average 65% increase in DHA (1.30% compared to 0.79%).

Plants homozygous for LBFLFK, QTL1, QTL2 and QTL3 were also field grown. The test plots contained three selections of a female parent containing the three QTL which have been described to confer the increase in EPA, DPA and DHA. Two control plots were included, which are of the same parental background but do not carry any of the three QTL. The field results confirm the results from the growth chamber (discussed above), where the addition of the three described genomic regions confer an increase in EPA, DPA and DHA in the field. The control plots averaged 9.440 EPA+DPA+DHA, while the plots with the three described QTL averaged 16.1740 EPA+DPA+DHA, and the highest plot contained 17.74% EPA+DPA+DHA. These three QTL have demonstrated an increase in EPA+DPA+DHA of up to 87.9% over the control, with an average 58% increase in EPA (11.08% compared to 7.03%), average 83% increase in DPA (3.47% compared to 1.90%), and average 65% increase in DHA (0.90% compared to 0.34%). All fatty acid analyses were performed by GC as described, using approximately 30 seeds subsampled from the full plot sample.

TABLE 7

Markers identifying the refined genomic

regions for QTL1, 2 and 3.

Size

QTL Position

QTL
(MB)
SNP Boundaries
in DH12075

QTL1: N1.1
3
Bn-A01-p9326695,
8879780-11922690

Bn-A01-p12510328

QTL2: N1.2
1.2
Bn-A01-p23330944,
22823086-24045492

Bn-A01-p24835449

QTL3: N6
1.7
Bn-A06-p17291868,
19156645-20846412

Bn-A06-p21415201

TABLE 8

Average PUFA values for Brassica

plants having the indicated QTL

QTL
EPA + DPA + DHA
EPA
DPA
DHA

N1.1, N1.2, N6
avg
20.04
14.32
4.41
1.30

stdev
1.74
1.34
0.51
0.26

n
17
17
17
17

max
22.95
16.74
5.45
1.87

N1.1, N1.2
avg
17.30
12.73
3.64
0.93

stdev
2.23
1.65
0.52
0.18

n
20
20
20
20

max
21.96
16.05
4.48
1.42

N6
avg
18.07
12.66
4.26
1.15

stdev
2.40
1.95
0.42
0.22

n
17
17
17
17

max
20.88
15.26
4.87
1.45

Control
avg
15.67
11.32
3.57
0.79

stdev
0.89
0.51
0.52
0.10

n
5
5
5
5

max
16.96
11.88
4.41
0.93

The region corresponding to each QTL was sequenced. The genomic sequence for each QTL was compared between the parent line with favourable alleles for increasing PUFA and the PUFA donor line, Kumily LBFLFK. Table 9 provides SNPs in chromosome N1 that were distributed across the QTL1 interval and Table 10 provides SNPs in chromosome N1 that are in candidate genes within QTL1. Table 11 provides SNPs in chromosome N1 that were distributed across the QTL2 interval and Table 12 provides SNPs in chromosome N1 that are in candidate genes within QTL2. Table 13 provides SNPs in chromosome N6 that were distributed across the QTL3 interval and Table 14 provides SNPs in chromosome N6 that are in candidate genes within QTL3. In each of these tables, “ref nt” refers to reference nucleotide in the PUFA donor line; “alt nt” refers to alternate nucleotide in the line with favourable alleles for increased PUFA; “AA change” refers to amino acid change; “type” refers to the polymorphism type; “TS” refers to transition; “TV” refers to transversion; and “subst” refers to substitution.

TABLE 9

SNPs in chromosome N1 distributed across the QTL1 interval

Ref
Alt

Codon
AA

Affected

Position
Nt
Nt
Sequence
Change
Change
Type
gene
Ortholog
Description

8,750,203
T
C
ATTTGAGAGTGGGAAA
TCT→
S ->P
TS
maker-
AT4G26170.1
| Symbols: | FUNCTIONS IN: molecular

AGACTTTGTAGGTAGTA
CCT

N1-

function unknown; INVOLVED IN:

GATACAGAGTCCAAGA

augustus-

biological process unknown; LOCATED

A[T/C]CTATCCAGGGTC

gene-

IN: chloroplast; EXPRESSED IN: 7 plant

TTGGTGTAGCTGTCAAC

8.369

structures; EXPRESSED DURING: F

ATTCATGACGCAGATGA

mature embryo stage, petal

CATT (SEQ ID NO: 1)

differentiation and expansion stage, E

expanded cotyledon stage, D bilateral

stage; BEST Arabidopsis thaliana protein

match is: effector of transcription2

(TAIR:AT5G56780.1); Has 75 Blast hits to

42 proteins in 17 species: Archae-0;

Bacteria-2; Metazoa-2; Fungi-0;

Plants-70; Viruses-0; Other

Eukaryotes-1 (source: NCBI BLink). |

chr4:13256930-13259382 FORWARD

LENGTH = 2453

8,844,910
C
T
CTGATACATTTAATAGA
CCG→
None
TS
maker-
AT4G26400.1
| Symbols: | RING/U-box superfamily

TTACCGTAAAACAAGTT
CCA

N1-

protein | chr4:13344808-13346597

TGCTGTACACCAGCTT[C/

augustus-

REVERSE LENGTH = 1790

T]GGAGGTGCTGAGAG

gene-

CACTTCTGCTTCTGCAG

8.434

AAACGGGAAGATAAAC

AAC (SEQ ID NO: 2)

8,952,616
G
A
TTTGTCGTCAACGTCGC
None
None
TS
maker-
AT4G26590.1
| Symbols: ATOPT5, OPT5 | oligopeptide

CTTCTTCCACATCCCCCA

N1-

transporter 5 | chr4:13413973-

TATAAACCTTAAACA[G/

augustus-

13417055 REVERSE LENGTH = 3083

A]CAACAAGAGATTTTT

gene-

AATGGAAACTGATGAA

8.442

GACTGATCTAATAGTCA

TA (SEQ ID NO: 3)

9,046,609
G
A
TCATTGCTAATGCTTTT
CAC→
None
TS
maker-
AT4G26760.1
| Symbols: MAP65-2 | microtubule-

GTTTTGAACACCATTTG
CAT

N1-

associated protein 65-2 |

CCTCATCTAAGCTCGG

fgenesh-

chr4:13478592-13481808 REVERSE

[G/A]TGAACTTCAGTGA

gene-

LENGTH = 3217

CCGTGGTCAAGAAATCT

9.391

AAACCAAGGACGGCGC

ATAG (SEQ ID NO: 4)

9,143,608
G
A
AGTTGGACCTGAGCTTA
TGC→
C→Y
TS
maker-
#N/A
#N/A

GTTTCCTTAAAGACGTG
TAC

N1-

GGACGGGCTGAGTACT

fgenesh-

[G/A]CAGGGTTACCGAT

gene-

GAAGAAGCCTTAGAAG

9.335

GTGCTCTACTACTTACTT

GGC (SEQ ID NO: 5)

9,248,592
C
T
ACATATCTTATTGGTTA
None
None
TS
None
None
None

TACATGATCCTCGTTTCT

GTCATCTTCATACTC[C/

T]GAAACAAAAAAATTA

AAAATCACCATATTAAT

TGCAAAGTTGTATCAAT

T (SEQ ID NO: 6)

9,347,120
G
A
CCGTTGAGAATCGTCCT
GAC→
None
TS
augustus_
AT4G27410.2
| Symbols: RD26, ANAC072 | NAC (No

GAGCGAATTCATCCGA
GAT

masked

Apical Meristem) domain transcriptional

GGAAGATCAAAAGACC

-N1-

regulator superfamily protein |

G[G/A]TCTTTCATCTCC

abinit-

chr4:13707240-13709149 REVERSE

GGGAACGAATCAAGAA

gene-

LENGTH = 1910

CGTCGTCGAGCTGAGA

9.68

CGAAGA (SEQ ID NO:

7)

9,454,361
C
T
CTCCCATCTGTCACTGC
GCA→
A→T
TS
maker-
AT4G27540.1
| Symbols: PRA1.H | prenylated RAB

AGTACTTGAACAGCTCC
ACA

N1-

acceptor 1.H | chr4:13753210-

CAAAGTGCTAAACTTG

fgenesh-

13754745 REVERSE LENGTH = 1536

[C/T]TAACAACCCCACCA

gene-

ATGCAAGTGGCATCTG

9.420

ATACCTACAAAGAAACA

CCA (SEQ ID NO: 8)

9,549,523
C
T
AACCATATCTACTCGTA
GAT→
D→N
TS
augustus_
AT4G27654.1
| Symbols: | unknown protein;

GGTTGGTCTTGATCGAT
AAT

masked-

FUNCTIONS IN: molecular function

TCTAGTTGAGAAGTAT

N1-

unknown; INVOLVED IN: biological

[C/T]GATCGGTGCCAGTT

abinit-

process unknown; LOCATED IN:

CACAAGTGTCGATCGAT

gene-

endomembrane system; EXPRESSED IN:

GATGTGCGTCTTTTTTT

9.111

17 plant structures; EXPRESSED

GC (SEQ ID NO: 9)

DURING: 9 growth stages; Has 30201

Blast hits to 17322 proteins in 780

species: Archae-12; Bacteria-1396;

Metazoa-17338; Fungi-3422; Plants-

5037; Viruses-0; Other Eukaryotes-

2996 (source: NCBI BLink). |

chr4:13811645-13812126 FORWARD

LENGTH = 482

9,652,028
T
G
TTCGCAGTGGTTCCGGT
GAT→
D→E
TV
augustus_
AT4G27750.1
| Symbols: ISI1 | binding |

GCTTTTTCGACATTCGTT
GAG

masked-

chr4:13841688-13843633 FORWARD

GAAAGAAGGGGAGGA

N1-

LENGTH = 1946

[T/G]GATAGAGTGACGA

abinit-

GCTTGGATCACATATTC

gene-

AGCGTTGAGCCGATGA

9.134

AGAT (SEQ ID NO: 10)

9,794,198
C
T
ACCAAACTCTGGATACG
GCT→
A→V
TS
maker-
AT4G28162.1
| Symbols: | Potential natural antisense

AAGCTTCTGGTCCAAGA
GTT

N1-

gene, locus overlaps with AT4G28160 |

TCCAGAGAAGATCAAG

augustus-

chr4:13980327-13980820 REVERSE

+C/T+TCCACCAGTGCCAC

gene-

LENGTH = 494

AACCATGACTTGAAAAC

9.249

AGTCAACAAATAAGCGT

TT (SEQ ID NO: 11)

9,847,417
A
G
ATTTCGATAGAGTCGCT
ATT→
I→V
TS
augustus_
AT4G28310.1
| Symbols: | unknown protein; BEST

GGAGAGAGCGACTCCA
GTT

masked-

Arabidopsis thaliana protein match is:

AAGCCATTAAGAAACC

N1-

unknown protein (TAIR:AT1G52270.1);

G[A/G]TTGAATCAAAGT

abinit-

Has 30201 Blast hits to 17322 proteins

CCATCGTGAAGGATAA

gene-

in 780 species: Archae-12; Bacteria-

GAAGAAGAAGACTGAG

9.179

1396; Metazoa-17338; Fungi-3422;

TCAAGC (SEQ ID NO:

Plants-5037; Viruses-0; Other

12)

Eukaryotes-2996 (source: NCBI BLink).

| chr4:14017408-14018208 FORWARD

LENGTH = 801

9,952,792
G
A
TGGGTAATCTCAGCCAC
GCC→
A→T
TS
maker-
AT5G47390.1
| Symbols: | myb-like transcription

TACTCGGGCTCTGGGTT
ACC

N1-

factor family protein | chr5:19226790-

GAGCGGGCTTGGCGGA

augustus-

19228858 FORWARD LENGTH = 2069

[G/A]CCGGGTCGAACA

gene-

ATCCTGGTTCTCCCGGT

9.258

GATGGCCATGACCACG

GCGTC (SEQ ID NO: 13)

10,052,015
A
C
ATAGTCAACAGCCGTCT
GTA→
None
TV
augustus_
AT2G31290.2
| Symbols: | Ubiquitin carboxyl-terminal

CAAGAACATCATCCTAA
GTC

masked-

hydrolase family protein |

GGTGTCCCAACCAGGT

N1-

chr2:13343846-13346070 FORWARD

[A/C]GCACCCGTTAAGTT

abinit-

LENGTH = 2225

TCTACAGAAGAAGTTCA

gene-

AGACTCTTGATCTCCAA

10.331

GG (SEQ ID NO: 14)

10,402,684
C
A
TGCATAAGTACTACTCG
GGG→
G→W
TV
maker-
AT4G16660.1
| Symbols: | heat shock protein 70 (Hsp

AACCCATGTCATAAAAT
TGG

N1-

70) family protein | chr4:9376737-

ATAACATGTCTCGACC

augustus-

9381507 FORWARD LENGTH = 4771

[C/A]ATTAGAGAAATCCT

gene-

TATCAATCCCATACTGC

10.157

AAAGCCGCACCGGAAT

GCT (SEQ ID NO: 15)

10,425,211
A
T
GAGCTCGAGTTCCTCAA
GCA→
None
TV
augustus_
AT2G47330.1
| Symbols: | P-loop containing

CCACGAACTAAACAGTA
GCT

masked-

nucleoside triphosphate hydrolases

GCGGGGAAGACAAAGC

N1-

superfamily protein | chr2:19428897-

[A/T]GAAGAACGTAAA

abinit-

19431720 REVERSE LENGTH = 2824

GGTCAAGCAGAAGCTG

gene-

AAGAAGACGAAGATGA

10.401

GAAGCC (SEQ ID NO:

16)

10,558,464
T
C
AAGTTTGGTGTTGACCT
CAT→
None
TS
maker-
AT4G16470.1
| Symbols: | Tetratricopeptide repeat

GTTAACATGTTCTAGCT
CAC

N1-

(TPR)-like superfamily protein |

TTCAGGTAGAGCATCA

fgenesh-

chr4:9287862-9289582 REVERSE

[T/C]AGAAAGAAAGAGC

gene-

LENGTH = 1721

AGCCTGACAAGACACTA

10.31

CAAGGTCTTTGCATCAC

TGG (SEQ ID NO: 17)

10,659,284
A
G
TCACAACCTAAGTCATA
ATT→
I→V
TS
augustus_
AT4G16350.1
| Symbols: CBL6, SCABP2 | calcineurin

TTCATATTACCAATTGC
GTT

masked-

B-like protein 6 | chr4:9242320-

AGTTACTGTGAATGAG

N1-

9243912 REVERSE LENGTH = 1593

[A/G]TTGAAGCTCTTTAC

abinit-

GAAATGTTCAAGAGCAT

gene-

CAGCAAAGACGGCCTT

10.449

ATC (SEQ ID NO: 18)

10,748,492
G
A
GACTATGCTGCTTTCGA
CCT→
P→S
TS
fgenesh_
AT3G22980.1
| Symbols: | Ribosomal protein

GAGTAAGAGCTTCACT
TCT

masked-

S5/Elongation factor G/III/V family

GTACAATGCTGAGTCA

N1-

protein | chr3:8160156-8163316

G[G/A]TGGTGTCTCAGT

abinit-

REVERSE LENGTH = 3161

ATGATCTTCTGTGAAGC

gene-

CAAGTCTGTGAGAGAC

10.205

ATGAG (SEQ ID NO: 19)

10,852,010
G
A
TCACCAAACTCACATGT
TTC→
None
TS
augustus_
#N/A
#N/A

CTGTTTCTTGACGTCTCT
TTT

masked-

TGCACACACTTTGTA[G/

N1-

A]AAGATTGCAATAATA

abinit-

TTAAGCTTTCCTTGTTCC

gene-

ACACGTTTCTTCATCAT

10.476

(SEQ ID NO: 20)

11,007,740
G
A
TTCCATCAGGAGGTTCT
GGT→
G→S
TS
augustus_
AT4G15810.1
| Symbols: | P-loop containing

GATGATTTTATGGTGCA
AGT

masked-

nucleoside triphosphate hydrolases

AAATGAGGGGAGTATA

N1-

superfamily protein | chr4:8989162-

[G/A]GTTCTAAGCAACT

abinit-

8992591 REVERSE LENGTH = 3430

CATCGAGGAGGATGGG

gene-

GGTGGTAGCATTCAAG

11.275

AATCT (SEQ ID NO: 21)

11,047,958
G
C
TTATCTCTAGCAAGTTA
AAG→
K→N
TV
maker-
#N/A
#N/A

CAATGAGATATTGGAT
AAC

N1-

GGAAGTGTTGATACTA

augustus-

A[G/C]TGTCTTATTGGT

gene-

ATATAATTTTGAACTGC

11.77

TTCTCTTAGATTATTTTT

ATC (SEQ ID NO: 22)

11,150,929
G
A
CTCTCCTCGCTTGGTGA
CCG→
P→S
TS
maker-
AT4G15560.1
| Symbols: CLA1, DEF, CLA, DXS, DXPS2 |

AACCGGAGAGACCATT
TCG

N1-

Deoxyxylulose-5-phosphate synthase |

GGTTTGCCTCATTGTCG

augustus-

chr4:8883907-8887565 FORWARD

[G/A]CATCTTTCCTCTTCT

gene-

LENGTH = 3659

ACCGGTAAGAATCTTGT

11.116

GAGGATAAGACTGATG

AC (SEQ ID NO: 23)

11,269,217
G
A
CCTGTCTCTAACATCTG
CGT→
R→C
TS
augustus_
#N/A
#N/A

CTGCAGAGTTACCCGCT
TGT

masked-

TCCACCATATCTCCAC[G/

N1-

A]GAGCCTGTACGCGA

abinit-

TTAGGCGAAGAGGACT

gene-

ATCTCTTGGTTCTTTCTT

11.325

GA (SEQ ID NO: 24)

11,343,118
G
C
TCCGAAACCTTGACCAA
GTG→
None
TV
augustus_
AT4G15390.1
| Symbols: | HXXXD-type acyl-

ATTCTACCCCCTCGCGG
GTC

masked-

transferase family protein |

GAAGAATCAACGGAGT

N1-

chr4:8792812-8794293 REVERSE

[G/C]ACTGTCGACTGTA

abinit-

LENGTH = 1482

ACGACGAAGGAGCTGT

gene-

TTTCGTCGACGCTCGTG

11.333

TCGA (SEQ ID NO: 25)

11,455,979
A
T
GGCTTGGTCTGGTAGG
TGT→
C→S
TV
maker-
AT4G15060.1
| Symbols: | CONTAINS InterPro

CATAATGAAAGCCGCTT
AGT

N1-

DOMAIN/s: FBD (InterPro:IPR013596), F-

GACATGTGTGATTAAAC

fgenesh-

box domain, Skp2-like

[A/T]AACAAAGTTCTGT

gene-

(InterPro:IPRO22364), FBD-like

ATGTCAAGGTATGTCGA

11.54

(InterPro:IPR006566), Leucine-rich

ATCGACTTCCATCTCCTC

repeat 2 (InterPro:IPR013101); BEST

CA (SEQ ID NO: 26)

Arabidopsis thaliana protein match is:

FBD, F-box and Leucine Rich Repeat

domains containing protein

(TAIR:AT1G55660.1); Has 30201 Blast

hits to 17322 proteins in 780 species:

Archae-12; Bacteria-1396; Metazoa-

17338; Fungi-3422; Plants-5037;

Viruses-0; Other Eukaryotes-2996

(source: NCBI BLink). | chr4:8599035-

8601761 FORWARD LENGTH = 2727

11,565,970
C
T
AAGAGCGATGGGCTTG
TCC→
None
TS
fgenesh_
AT4G14920.1
| Symbols: | Acyl-CoA N-acyltransferase

GTGACTTTTCTTCCTTTC
TCT

masked-

with RING/FYVE/PHD-type zinc finger

CGGAGACGAGTGATTC

N1-

protein | chr4:8531043-8535842

[C/T]GAGTCTAGCGATA

abinit-

REVERSE LENGTH = 4800

AGCCCCTCATGGCACAC

gene-

TATGGCAATGTAGAAG

11.201

AGCC (SEQ ID NO: 27)

11,659,776
C
G
GATCTGTCGAGCTTAGT
AAG→
K→N
TV
maker-
AT4G14750.1
| Symbols: |QD19 | IQ-domain 19 |

CGCTATCCACGGATAAT
AAC

N1-

chr4:8470381-8472187 FORWARD

ACTCATAATGACTTTC[C/

augustus-

LENGTH = 1807

G]TTTCCTAATTGAGAT

gene-

GAAGCTCTATGCACCCG

11.142

TACGGCCTTAAGTACAC

C (SEQ ID NO: 28)

11,726,807
C
T
GTGTATATCGTTAACCC
CTG→
None
TS
maker-
AT4G14730.1
| Symbols: | Bax inhibitor-1 family

CACGACCATTGTAGCTG
CTA

N1-

protein | chr4:8448549-8450073

TGAGAACCGCAGCCTC

augustus-

FORWARD LENGTH = 1525

[C/T]AGAACAATCTTCCC

gene-

TGTTTTTTTTATTCACAA

11.144

GAGTTAGATTGTAACAT

C (SEQ ID NO: 29)

11,850,103
A
G
AAGCACTCGACTTATAA
CTG→
L→P
TS
augustus_
AT4G25000.1
| Symbols: ATAMY1, AMY1 | alpha-

CCATGTTTGATATGTTT
CCG

masked-

amylase-like | chr4:12851969-12853845

CGACGATCTACCAGTC

N1-

REVERSE LENGTH = 1877

[A/G]GCTTCTGTGAAGT

abinit-

CTGCTCAGCAACGTGGT

gene-

CAGCCCTTTCATGACCA

11.410

CTC (SEQ ID NO: 30)

11,956,477
C
T
CACCATCACCAGCTATG
CCA→
P→S
TS
augustus_
AT4G14580.1
| Symbols: CIPK4, SnRK3.3 | CBL-

GGCTTTCCATCTCCACC
TCA

masked-

interacting protein kinase 4 |

ATCACCAGAAAAAGCC

N1-

chr4:8367887-8369167 REVERSE

[C/T]CAGGGACCATTCTG

abinit-

LENGTH = 1281

CTCGGTAAATACGAACT

gene-

CGGTCGCCGATTAGGC

11.420

AGT (SEQ ID NO: 31)

TABLE 10

SNPs in chromosome N1 that are in candidate genes within QTL1

Ref
Alt

Codon
AA

Affected

Position
Nt
Nt
Sequence
Change
Change
Type
gene
Ortholog
Description

9,136,686
A
C
TAGTTGAGAAACGTTTTG
AAG→
K→Q
TV
augustus_
AT4G27030.1
| Symbols: FAD4, FADA | fatty acid

TGAGTCCTCCTCTTTCCAA
CAG

masked-

desaturase A | chr4:13571921-

CGACCCAACTCTG[A/C]A

N1-

13573070 FORWARD LENGTH = 1150

GTCTACATGGACTCACCG

abinit-

CTTATGGGTTGCAGCTGG

gene-

TTGCACCACCTTG (SEQ

9.32

ID NO: 32)

9,641,936
G
A
TATTGATAAGTATGGATT
CCT→
P→L
TS
maker-
AT5G42870.2
| Symbols: PAH2 | phosphatidic acid

CGAGTACCAATTCAATGG
CTT

N1-

phosphohydrolase 2 | chr5:1718547

TAAAAAAGATGTCA[G/A]

augustus-

17189943 REVERSE LENGTH = 4466

GTCTTACCTGCTTTAGATT

gene-

ATTTAAGAAACTTCTTGTT

9.307

AGATATGCCTGA (SEQ ID

NO: 33)

10,613,015
C
G
TTTAAACAGGTTGCTTGA
CAG→
Q→E
TV
maker-
AT4G16440.1
| Symbols: | ferredoxin hydrogenase

ACGGTGGTGGGCAGATTA
GAG

N1-

| chr4:9269094-9271669 REVERSE

AGCCAAAAACGGGA[C/G]

augustus-

LENGTH = 2576

AGACTCCGAAAGAACTGA

gene-

TCAACTCACTTGAAGCTAC

10.123

TTATATGAATGAT (SEQ ID

NO: 34)

9,040,901
A
G
AGGAGGATGAGAATTCGT
AAT→
None
TS
maker-
AT5G55240.1
| Symbols: ATPXG2 | ARABIDOPSIS

GTTACCGGAAGAGTAGCA
AAC

N1-

THALIANA PEROXYGENASE 2 |

TAGCTAAGGGCCAA[A/G]

fgenesh-

chr5:22405926-22407351 FORWARD

TTGATTACAGCGGCTGCG

gene-

LENGTH = 1426

ATAAGCGATACAATGATA

9.389

TTGAAACCAAGCAT (SEQ

ID NO: 35)

9,048,617
C
A
TATATTAATCCAGGACATT
None
None
TV
maker-
AT4G26770.1
| Symbols: | Phosphatidate

GGCATTGTAAGAAAAATC

N1-

cytidylyltransferase family protein |

ATTTTCAAAAGGG[C/A]A

augustus-

chr4:13482074-13484849 FORWARD

ATGGAGAAAGATCTGAAT

gene-

LENGTH = 2776

CAGAACTCTCCACGAATC

9.206

AGAAAGCTAAGGG (SEQ

ID NO: 36)

9,352,326
A
G
AGCAAATACATATTATATT
TTT→
F→S
TS
augustus_
AT4G27420.1
| Symbols: | ABC-2 type transporter

TATATATTAAAATACCTGA
TCT

masked-

family protein | chr4:13712199-

TCTTGTAAGTGA[A/G]AA

N1-

13714797 REVERSE LENGTH = 2599

AGGTCCGTTTTCCACCAA

abinit-

AGGAGACCACATAAGAAA

gene-

GACACGACAAAA (SEQ ID

9.69

NO: 37)

9,921,975
TG
GA
GGATATGATTGAGACAGG
ATG→
M→R
TV
maker-
AT4G17480.1
| Symbols: | alpha/beta-Hydrolases

GATTAATGATCAATGCTC
AGA

N1-

superfamily protein | chr4:9745006

AAGCGTTGAAGCTA[TG/

augustus-

9746966 REVERSE LENGTH = 1961

GA]AGCTTCACACAGCTCCT

gene-

TAGGAACCTCTCCAGCTC

9.257

CCCTGGCTACTGCTT (SEQ

ID NO: 38)

10,706,805
G
T
AAAGCAATCGACTCAGCA
GAC→
D→E
TV
maker-
AT1G54350.1
| Symbols: | ABC transporter family

TTTTCCCTGACGCGGACA
GAA

N1-

protein | chr1:20286882-20290401

AGGTTGTAACGAAA[G/T]

augustus-

FORWARD LENGTH = 3520

TCAGCCTCTTTCTTCTCTT

gene-

GCATAAAGTTGAGATTCA

10.169

CTAGTCCCTGAGA (SEQ

ID NO: 39)

TABLE 11

SNPs in chromosome N1 distributed across the QTL2 interval

Ref
Alt

Codon
AA

Affected

Position
Nt
Nt
Sequence
Change
Change
Type
gene
Ortholog
Description

22,823,086
T
C
CACCATTTCTAGATC
None
None
TS
maker-
AT3G17000.1
| Symbols: UBC32 | ubiquitin-

TATAGAATCAGAATT

N1-

conjugating enzyme 32 | chr3:5797179-

CGAATTGGATCTTCG

augustus-

5799683 FORWARD LENGTH = 2505

GTGAA[T/C]CTAATC

gene-

TCCAATATCATCTCC

22.349

TCTGATCTCTAAAGC

TTGAGGAGGATAAC

(SEQ ID NO: 40)

22,880,595
A
C
ATAGAGTAACGATT
GTT→
None
TV
maker-
AT3G16830.1
| Symbols: TPR2 | TOPLESS-related 2 |

ATCATCAACCTTAGT
GTG

N1-

chr3:5731534-5737772 FORWARD

GAACCCTGAGAGAT

fgenesh-

LENGTH = 6239

ACTTCTC[A/C]ACTT

gene-

CGTCCCATTCACCGG

22.246

CGAGAGCTTTCTCTT

CAAAGTACTTTATAT

T (SEQ ID NO: 41)

22,902,670
G
A
ATGTTTATTCATTTTT
None
None
TS
None
None
None

CATAAGTTTTTTTTA

GTTTTCATTTACATTT

TAT[G/A]TATGATTT

TATCAAACTACATAA

ATAAAAAATCTACAA

ACTTTAATCAAA

(SEQ ID NO: 42)

22,949,738
C
T
CATTATCCTGAAGCA
ACG→
None
TS
maker-
AT3G16710.1
| Symbols: | Pentatricopeptide repeat

ATGAGTTTCTAACCT
ACA

N1-

(PPR) superfamily protein I

GTAAATATACTTATA

augustus-

chr3:5690020-5691543 FORWARD

ACCTG[C/T]GTGTCC

gene-

LENGTH = 1524

TCTCCCCCAAAACTG

22.357

CAACTGAGCCATCAT

ATGCTTGAGATCAA

(SEQ ID NO: 43)

23,011,207
C
T
TCCTCCCACATAGGT
GTC→
None
TS
maker-
AT3G16500.1
| Symbols: PAP1, IAA26 | phytochrome-

CAAATCAATAAGAA
GTT

N1-

associated protein 1 | chr3:5612500-

TGGTGATGGTGTGA

augustus-

5614410 REVERSE LENGTH = 1911

AGCAAGT[C/T]GAAC

gene-

CTAAGAGGGAAGGC

23.126

ATGTTTGTAAAGATC

AACATGGACAGTGT

TCC (SEQ ID NO: 44)

23,044,228
C
G
CTATATTTTATAACA
None
None
TV
None
None
None

ACAACTTTCGCAATA

TCATATTTTTATGGA

TTAAT[C/G]ATGTGG

TAATAAAAATTCTAG

TTCTCCCAAATTAAG

GTACAAAATTAATG

(SEQ ID NO: 45)

23,099,592
A
G
CAGCAAGCCAGCCT
ATG→
M→V
TS
maker-
AT3G16250.1
| Symbols: NDF4 | NDH-dependent

CCAGTCGCAGATGA
GTG

N1-

cyclic electron flow 1 | chr3:5506931-

GCCGAATGATGAAC

augustus-

5508414 REVERSE LENGTH = 1484

CTCCTGCT[A/G]TGG

gene-

ATTTTGCGTTCGTCC

23.129

ATGTAAGTAGTGAT

CTTCACTCGCTAGTT

GCT (SEQ ID NO: 46)

23,176,771
A
G
CTCACAGAGCTATAC
TTT→
None
TS
maker-
AT3G16090.1
| Symbols: | RING/U-box superfamily

ATAAACAAACCATCC
TTC

N1-

protein | chr3:5456144-5458966

ACAACAAGAAGAAA

fgenesh-

FORWARD LENGTH = 2823

CGCCAT[A/G]AACG

gene-

AAACAATACGAACG

23.75

TGCGCGAGCTTAGA

GACGTTAGGAGTCG

TCTC (SEQ ID NO:

47)

23,201,595
G
A
TGATAAGGTTACTGC
AGT→
S→N
TS
maker-
AT3G16000.1
| Symbols: MFP1 | MAR binding

AAAGAAAGTTGTCA
AAT

N1-

filament-like protein 1 | chr3:5430889-

GGAGGAGAAAGAG

fgenesh-

5433817 REVERSE LENGTH = 2929

CAGTACTA[G/A]TTC

gene-

TTGAGGAGACAAAG

23.12

GTTATAATCACCAGT

TCTTGCTCCAGTTAT

TAT (SEQ ID NO: 48)

23,257,618
A
C
AGATAATGTGACTG
GAT→
D→A
TV
maker-
AT3G15920.1
| Symbols: | Phox (PX) domain-

ATTGGCATGAACTA
GCT

N1-

containing protein | chr3:5383596-

ATCACAGAATCTGG

augustus-

5387009 REVERSE LENGTH = 3414

ACTTCTTG[A/C]TAA

gene-

GAGTCATTTTACTGA

23.138

TAGAGCTGCAGAAA

CCGGAGAGGCATCA

ATAT (SEQ ID NO:

49)

23,302,268
G
A
CTGGAGCATGTACTC
CTA→
None
TS
maker-
AT3G15830.1
| Symbols: | phosphatidic acid

CTCTACGCCCGTGAA
TTA

N1-

phosphatase-related/PAP2-related |

GAACAGAACTCCTA

augustus-

chr3:5354974-5356424 FORWARD

CCGCTA[G/A]CAAAC

gene-

LENGTH = 1451

ACGGTATCCAATGG

23.199

ACTCTCACCACGTGG

ACAGGATTGCACAT

CC (SEQ ID NO: 50)

23,367,822
T
C
GACTCAGTGAGAGC
TTT→
None
TS
maker-
AT1G28030.1
| Symbols: | 2-oxoglutarate (2OG) and

CCAAGTCCGAAAAG
TTC

N1-

Fe(II)-dependent oxygenase superfamily

CCCTAGAAGAGTAC

augustus-

protein | chr1:9771793-9773345

GGATGTTT[T/C]GAG

gene-

FORWARD LENGTH = 1553

GCCACGTTCGATGG

23.144

AGTTTCAGCGGAGC

TAAGGAAGGCTATT

TTCAA (SEQ ID NO:

51)

23,380,089
C
A
TCTAGGTTGCAGCCC
GTG→
V→L
TV
augustus_
AT3G15540.1
| Symbols: |AA19, MSG2 | indole-3-

AAATCCGGTAGCCTC
TTG

masked-

acetic acid inducible 19 | chr3:5264024-

GGATCTTTTCATAAT

N1-

5265678 FORWARD LENGTH = 1655

CCTCA[C/A]CCTCCT

abinit-

GCATGACTCTATGAA

gene-

CATTCTGCAAAATCA

23.488

AAATACCCAACCCA

(SEQ ID NO: 177)

23,457,696
A
C
TGATCAGGCTTCTCT
AAT→
N→K
TV
augustus_
AT3G15400.2
| Symbols: ATA20 | anther 20 |

ATGTCATCAATCTTC
AAG

masked-

chr3:5201644-5203197 FORWARD

TCTTTACCACCATCA

N1-

LENGTH = 1554

TTAAG[A/C]TTCTCT

abinit-

TTGTAATCAGGCATT

gene-

ACTTTATGAGCATGC

23.498

TTTTTTTCAGGATC

(SEQ ID NO: 52)

23,520,607
G
A
AAAGAAGAGCTTAG
CTC→
L→F
TS
augustus_
#N/A
#N/A

TCAACGCATTGAAG
TTC

masked-

AAAGATAGTCCGGT

N1-

GGGCCAGA[G/A]TC

abinit-

TTGTTCTTTGTTTGA

gene-

ATTTGGCATTACCGG

23.516

GAAAGGCATGTGAG

TGTG (SEQ ID NO:

53)

23,552,773
C
T
ACAGAACTGGTATT
CCG→
P→S
TS
maker-
AT3G15115.1
| Symbols: | unknown protein; BEST

AATAGTAGTTGTAAT
TCG

N1-

Arabidopsis thaliana protein match is:

AACAATAGTGTCCA

augustus-

unknown protein (TAIR:AT1G53180.1);

GTGTTGT[C/T]CGAT

gene-

Has 47 Blast hits to 47 proteins in 15

GGGAGGGAGTTTAC

23.153

species: Archae-0; Bacteria-0;

AGAGAACTCAGACG

Metazoa-13; Fungi-0; Plants-30;

TTACCTAGTTACATA

Viruses-0; Other Eukaryotes-4

GGA (SEQ ID NO:

(source: NCBI BLink). | chr3:5085992-

54)

5087489 REVERSE LENGTH = 1498

23,598,941
A
G
CGGGGAGGACGGG
CTC→
L→A
subst
augustus_
AT3G14920.1
| Symbols: | Peptide-N4-(N-acetyl-beta-

AGGCTTTTTGACCTC
GCC

masked-

glucosaminypasparagine amidase A

GAGGTAGGGAGTAG

N1-

protein | chr3:5018275-5020273

GTGAGGTG[A/G]GG

abinit-

FORWARD LENGTH = 1999

TTTTGGGATGGAGG

gene-

AGAGATGAGGAGA

23.533

GGGGAGAAATGTGG

TGGTTTG (SEQ ID

NO: 55)

23,670,623
T
C
TGTGGCTTTGGGAA
CCA→
None
TS
maker-
AT3G14640.1
| Symbols: CYP72A10 | cytochrome

ATCATAAACGTTGTT
CCG

N1-

P450, family 72, subfamily A,

GAATACTTCTTTGAT

augustus-

polypeptide 10 | chr3:4919856-4921787

TTGCTC[T/C]GGATC

gene-

FORWARD LENGTH = 1932

CATTATGGTGATGGT

23.230

TGGTATAGGTCCAA

ACCATGTATAGTAA

GT (SEQ ID NO: 56)

23,682,848
C
A
TGACGAGAGCACAG
CGA→
R→L
TV
maker-
AT3G59550.1
| Symbols: SYN3, ATRAD21.2, ATSYN3 |

TCACGGAACAGATA
CTA

N1-

Rad21/Rec8-like family protein |

ATCAACTTGCTTTGA

augustus-

chr3:21997054-22000678 FORWARD

ATAGATT[C/A]GGAC

gene-

LENGTH = 3625

AACACCAAGTAAAA

23.231

GATGACCAGACAAT

CTCAATGCCAAGGT

GCCT (SEQ ID NO:

57)

23,745,365
T
A
GGGATGCTAGGAAT
CAT→
H→Q
TV
maker-
AT3G14490.1
| Symbols: | Terpenoid cyclases/Protein

GTTTTTCGAGCCACG
CAA

N1-

prenyltransferases superfamily protein |

ATATTCACTTGGAAG

fgenesh-

chr3:4863631-4865949 REVERSE

AATTCA[T/A]ACCGT

gene-

LENGTH = 2319

TAAACTTACAATGGT

23.43

CCTCACTGTTGTGGA

TGATACATGTGATGC

(SEQ ID NO: 58)

23,792,572
C
A
TCAATGGCGGCGGA
ACC→
None
TV
fgenesh_
AT3G14410.1
| Symbols: | Nucleotide/sugar

TCGGAGCAAAGGCG
ACA

masked-

transporter family protein |

TTGTGAGAGACGAG

N1-

chr3:4815811-4817980 REVERSE

TTCGTGAC[C/A]TAT

abinit-

LENGTH = 2170

GCTTACATTCTTCTCT

gene-

ACATCGCTCTCTCTA

23.282

GCGGTCAAATCTTCT

T (SEQ ID NO: 59)

23,855,829
G
A
CGGAACGGTCGACT
TGC→
C→Y
TS
fgenesh_
AT3G14310.1
| Symbols: ATPME3, PME3 | pectin

TCATCTTCGGAAACG
TAC

masked-

methylesterase 3 | chr3:4771902-

CCGCTGTCGTTCTCC

N1-

4775119 REVERSE LENGTH = 3218

AAAACT[G/A]CGAC

abinit-

ATCCACGCTCGCCGA

gene-

CCAAACTCCGGCCA

23.288

GAAAAACATTGTCA

CGG (SEQ ID NO:

60)

23,910,029
T
C
ATAGAGTCCGGTGG
AAT→
None
TS
maker-
AT3G14230.3
| Symbols: RAP2.2 | related to AP2 2 |

TCAAGCTGAGAAGT
AAC

N1-

chr3:4737146-4739136 REVERSE

CTGCTAAGAGAAAG

augustus-

LENGTH = 1991

AGAAAGAA[T/C]CA

gene-

GTACAGGGGGATTA

23.175

GGCAGCGACCTTGG

GGAAAATGGGCTGC

TGAGAT (SEQ ID

NO: 61)

23,947,522
C
T
ACGACGACCAGATC
CCG→
P→S
TS
maker-
AT3G14130.1
| Symbols: | Aldolase-type TIM barrel

CGCTCTCACTCCGAC
TCG

N1-

family protein | chr3:4685646-4688309

TCACCCGATCCGTCT

augustus-

REVERSE LENGTH = 2664

TCTTCT[C/T]CGCCG

gene-

CCGCCGTCGGGAAA

23.179

AGTCACGGTAACGG

TGGCTTTCCCAGGTC

CG (SEQ ID NO: 62)

24,021,883
A
C
GAGAATCTTGAAAG
GTT→
V→G
TV
maker-
AT3G13960.1
| Symbols: AtGRF5, GRF5 | growth-

CTTCTAGAAGCTTCT
GGT

N1-

regulating factor 5 | chr3:4608383-

GGAAAAAAAGTTCT

augustus-

4610399 FORWARD LENGTH = 2017

TTCCCCA[A/C]CTCC

gene-

AACTCTCTCCCCAAT

24.75

CCCTTGAAAATATCT

ACAATTAATCATAAT

T (SEQ ID NO: 63)

24,056,999
G
A
CACAGAACGCTAAC
TCC→
S→F
TS
fgenesh_
AT3G13880.1
| Symbols: | Tetratricopeptide repeat

GCGCCAGCGTAAGT
TTC

masked-

(TPR)-like superfamily protein |

GAACTTATCGAGCTT

N1-

chr3:4572180-4574490 FORWARD

CAAATTG[G/A]AATC

abinit-

LENGTH = 2311

TCTAGCTTCTAGGAA

gene-

CAGCTTCATGGAGT

24.382

GTTCGTGAAACCCA

ACT (SEQ ID NO: 64)

24,111,397
C
T
GGCAGAGACTAAAG
GCA→
A→T
TS
maker-
AT2G28470.2
| Symbols: BGAL8 | beta-galactosidase 8

AGACAAGAAACCCT
ACA

N1-

| chr2:12168915-12173679 REVERSE

AGAAGAAACAGAGC

augustus-

LENGTH = 4765

AGAAGCTG[C/T]TGC

gene-

CATAGCCACATTCTT

24.79

CATTACTGTTGAAAG

CAAAATATCTTGCTT

TA (SEQ ID NO: 65)

24,153,185
A
T
TAGTAGTGAATGCG
TAT→
Y→F
TV
augustus_
AT3G13682.1
| Symbols: LDL2 | LSD1-like2 |

AGCATTTGATCTCCG
TTT

masked-

chr3:4479193-4481509 REVERSE

CTGCTTATGAGTTTC

N1-

LENGTH = 2317

TGTTGT[A/T]TAATG

abinit-

GTTTCATTAACTTTG

gene-

GGGTGTCTCCGTTGT

24.481

TTAATGGATATGTTC

(SEQ ID NO: 66)

24,207,903
C
T
CGATGAAATTTGATC
CCA→
P→S
TS
augustus_
AT3G13680.1
| Symbols: | F-box and associated

TTCAAGGAATTGGA
TCA

masked-

interaction domains-containing protein

AACGAAAGAGACTT

N1-

| chr3:4477289-4478721 REVERSE

CATTGAT[C/T]CATC

abinit-

LENGTH = 1433

TATAAAGCAAGTAA

gene-

GTATACTTGACCAAG

24.492

TAGAGATCACTCAA

GTA (SEQ ID NO: 67)

24,242,389
G
T
GGAGGAGGGAAGA
AAG→
K→N
TV
augustus_
AT3G13360.1
| Symbols: WIP3 | WPP domain

TTGATTCAGGTAGCC
AAT

masked-

interacting protein 3 | chr3:4338359-

AAGGGAGAGATACC

N1-

4340412 REVERSE LENGTH = 2054

ATCAAGAA[G/T]GG

abinit-

GAGTGAAGAGCGCA

gene-

TAGATTCGGACTTGA

24.500

GAAGTAGCGATTTT

GTGTT (SEQ ID NO:

68)

24,304,491
G
C
GCTACGTTGCGCCGT
GAA→
E→Q
TV
maker-
AT3G13080.4
| Symbols: ATM RP3, MRP3, ABCC3 |

ATCTCATGGACAGTT
CAA

N1-

multidrug resistance-associated protein

TTGTTCAGTACCTGA

augustus-

3 | chr3:4195799-4201265 REVERSE

ACGGT[G/C]AAAGG

gene-

LENGTH = 5467

CAATACAAATACCAA

24.24

GGATACGTTTTGGTA

ACGATTTTCTTCGTT

(SEQ ID NO: 69)

24,359,101
T
G
CTCAATGATGAAGA
ATC→
I→S
TV
augustus_
AT3G12955.1
| Symbols: | SAUR-like auxin-responsive

AGATGAGAATGATG
AGC

masked-

protein family | chr3:4135500-4136143

ATGCTGTTAAGGAG

N1-

REVERSE LENGTH = 644

ATGCAAGA[T/G]CG

abinit-

TGTCCACACAATTAA

gene-

GTCGTTCATATTCCT

24.530

ACACAAGCCTCAGA

TCCC (SEQ ID NO:

70)

24,362,374
T
A
ATCATATGGGGAGG
GGT→
None
TV
augustus_
AT3G12950.1
| Symbols: | Trypsin family protein |

CACTGGTAGCCGTG
GGA

masked-

chr3:4132528-4135134 REVERSE

GGAGGTTGAAGCTG

N1-

LENGTH = 2607

AAAGTAGG[T/A]GA

abinit-

GTCTCCAGAGAGCT

gene-

GGACTACTGGAGTT

24.531

GATCTGGGAAGGTT

ACTTAC (SEQ ID NO:

71)

24,456,688
A
C
AAAGCTGCTTCGATC
AAT→
N→K
TV
augustus_
AT3G12760.1
| Symbols: | CONTAINS InterPro

TGCCAATCACTGGCT
AAG

masked-

DOMAIN/s: Defective-in-cullin

TTAAGAGCCTGAAG

N1-

neddylation protein

AGCACT[A/C]TTCTC

abinit-

(InterPro:IPR014764), Protein of

ACTGCTCCCCCAGCA

gene-

unknown function DUF298

CATTATTAAACAAAT

24.551

(InterPro:IPR005176), UBA-like

CACACACACAAAAA

(InterPro:IPR009060); BEST Arabidopsis

A (SEQ ID NO: 72)

thaliana protein match is: Domain of

unknown function(DUF298)

(TAIR:AT1G15860.2); Has 857 Blast hits

to 855 proteins in 202 species: Archae-

0; Bacteria-0; Metazoa-482; Fungi-

154; Plants-139; Viruses-0; Other

Eukaryotes-82 (source: NCBI BLink). |

chr3:4054739-4056980 FORWARD

LENGTH = 2242

24,500,169
C
T
GTAAATAAAGAGTA
AGG→
RVK
TS
maker-
AT3G12640.1
| Symbols: | RNA binding

ACAATCATTACATTG
AAG

N1-

(RRM/RBD/RNP motifs) family protein |

GCGACAAAGATTGT

augustus-

chr3:4014213-4017869 FORWARD

TCGAGAC[C/T]TAGC

gene-

LENGTH = 3657

ATCTTCCAAAGGGC

24.103

GGCTAGTAGATAAT

GTCCCTACAAATACC

AGT (SEQ ID NO: 73)

TABLE 12

SNPs in chromosome N1 that are in candidate genes within QTL2

Ref
Alt

Codon
AA

Affected

Position
nt
nt
Sequence
Change
Change
Type
gene
Ortholog
Description

23,089,542
C
G
CTTGAAAACGTAGA
GAC→
D→H
TV
augustus_
AT3G16340.2
| Symbols: PDR1 | pleiotropic drug

AGAAAGCGTTGCG
CAC

masked-

resistance 1 | chr3:5539788-5546449

TTTTATGAGAAGCA

N1-

FORWARD LENGTH = 6662

GTTCTCTGT[C/G]C

abinit-

CAACAGATCTTGAA

gene-

GAGGTCAGATTTG

23.422

GGGACAGAGTGTT

TGTTGAATA (SEQ

ID NO: 74)

23,089,635
A
T
GTTGAATACGAGA
TCC→
S→T
TV
augustus_
AT3G16340.2
| Symbols: PDR1 | pleiotropic drug

GAAGCTGGATGTG
ACC

masked-

resistance 1 | chr3:5539788-5546449

ATTTGAATCTGTCG

N1-

FORWARD LENGTH = 6662

TAAGGGACGG[A/T]

abinit-

TAGTTCGTTTTCAA

gene-

GATTGGCACCGACA

23.422

TGGAAGGTTTTGG

ACTGTTTGG (SEQ

ID NO: 75)

23,090,743
T
G
AATGGGGAAAACT
ATT→
I→L
TV
augustus_
AT3G16340.2
| Symbols: PDR1 | pleiotropic drug

AGACCTGTTGTGAC
CTT

masked-

resistance 1 | chr3:5539788-5546449

TCTTTTCTTTTGACC

N1-

FORWARD LENGTH = 6662

ACCTGAAA[T/G]TC

abinit-

CTCGTGTCATTTCG

gene-

TCTCCGACCATCGT

23.422

ATCTTTGCATAAGT

CAAGCC (SEQ ID

NO: 76)

23,090,785
A
T
ACCTGAAATTCCTC
TTA→
L→I
TV
augustus_
AT3G16340.2
| Symbols: PDR1 | pleiotropic drug

GTGTCATTTCGTCT
ATA

masked-

resistance 1 | chr3:5539788-5546449

CCGACCATCGTATC

N1-

FORWARD LENGTH = 6662

TTTGCATA[A/T]GT

abinit-

CAAGCCCTAATATC

gene-

TAAAATAGAAAATG

23.422

ATCAATAGTTGCCC

AAATTT (SEQ ID

NO: 77)

23,091,367
T
C
GTTAGAGATATTAA
TAC→
Y→C
TS
augustus_
AT3G16340.2
| Symbols: PDR1 | pleiotropic drug

ATCTAGGTTAAAAG
TGC

masked-

resistance 1 | chr3:5539788-5546449

TGAGATCATTACTT

N1-

FORWARD LENGTH = 6662

TGAGAGTG[T/C]AG

abinit-

TCTGTGATGAGACT

gene-

GCTCTTGACATTCC

23.422

CAGCTGCGATGGA

CTTCATG (SEQ ID

NO: 78)

23,092,042
A
G
GAGTTAATTACCTT
ATT→
I→T
TS
augustus_
AT3G16340.2
| Symbols: PDR1 | pleiotropic drug

GAGGGTTTCATGAT
ACT

masked-

resistance 1 | chr3:5539788-5546449

ACCAGAAGCTTCTC

N1-

FORWARD LENGTH = 6662

TAAGAATA[A/G]TG

abinit-

AGTTTAGTGGTTTT

gene-

TGCAAAGTTTAAAC

23.422

CAAATAAACGAAG

GCCTCTC (SEQ ID

NO: 79)

23,150,402
C
G
GAGTGCAAGTGGA
GGA→
G→R
TV
maker-
AT3G16170.1
| Symbols: | AMP-dependent synthetase

ACTGCTACGCCACC
CGA

N1-

and ligase family protein | chr3:5476074-

AGTAAACCAAGTCC

augustus-

5480302 FORWARD LENGTH = 4229

CAAGGACTC[C/G]T

gene-

GCAACAAACTCAGC

23.190

TGATGGTTTCGCCA

CAATTCCAACTCTA

GCTCCTT (SEQ ID

NO: 80)

23,150,595
TC
CA
GGATAGGTTAGAA
GAT→
D→C
Subst
maker-
AT3G16170.1
| Symbols: | AMP-dependent synthetase

GTAGAAATAGCAA
TGT

N1-

and ligase family protein | chr3:5476074-

CAAAAATTCAAACA

augustus-

5480302 FORWARD LENGTH = 4229

GATCACCATA[TC/CA]

gene-

TACATTTTTAGTA

23.190

TCTTCCTTGTGGAA

CAACTTAGAGATGG

TAAAGGCAG (SEQ

ID NO: 81)

23,155,220
G
T
CCACAAACCACCTC
GAC→
D→E
TV
maker-
AT3G16150.1
| Symbols: | N-terminal nucleophile

TCCTTTATTCGACA
GAA

N1-

aminohydrolases (Ntn hydrolases)

CGGCAATGAGTCCA

augustus-

superfamily protein | chr3:5471735-

GCGAACCC[G/T]TC

gene-

5473276 FORWARD LENGTH = 1542

GTCGAGCCGATGCT

23.191

TGATGACGTAATCA

ACCGCTTCTTGTAG

CCCAAC (SEQ ID

NO: 82)

23,155,766
C
A
GCTTCTTTGGCCAG
GAG→
E→D
TV
maker-
AT3G16150.1
| Symbols: | N-terminal nucleophile

CTTGAGCATCCCCA
GAT

N1-

aminohydrolases (Ntn hydrolases)

CGTTGTCTTCCGTG

augustus-

superfamily protein | chr3:5471735-

ACGAAGTA[C/A]TC

gene-

5473276 FORWARD LENGTH = 1542

GTTGTCCACAGTTT

23.191

CAACTCCCTAATAA

AAACCAAATCGATT

TCGGTT (SEQ ID

NO: 83)

23,314,197
T
C
CAGCAAGTGACCA
AAC→
N→D
TS
augustus_
AT3G15730.1
| Symbols: PLDALPHA1, PLD |

GGAAGGTCATGTTC
GAC

masked-

phospholipase D alpha 1 | chr3:5330322-

CAGTGACTCACTTG

N1-

5333745 FORWARD LENGTH = 3424

AGTAAAAGT[T/C]C

abinit-

CAGTACTTATCAGC

gene-

AATACGGTTAACTT

23.470

TCTCAATGCATTCC

AAGCTTG (SEQ ID

NO: 84)

23,318,357
C
T
TTGTAGATCGATCG
GTT→
V→I
TS
augustus_
AT3G15730.1
| Symbols: PLDALPHA1, PLD |

TTGCGTAGAGCTGT
ATT

masked-

phospholipase D alpha 1 | chr3:5330322-

GTTTCTCCTTTGCC

N1-

5333745 FORWARD LENGTH = 3424

GAAACCAA[C/T]TG

abinit-

CCTCTTCAACATTT

gene-

GCTCTAATCTGGAA

23.470

TTTAAAACAGAGGT

TAAAGA (SEQ ID

NO: 85)

23,343,089
C
T
TGGTGCTTTCCTTT
AGG→
None
TS
fgenesh_
AT3G15650.1
| Symbols: | alpha/beta-Hydrolases

AGGCCTCACAACAT
AGA

masked-

superfamily protein | chr3:5305926-

AAGTCCTTCCAAAC

N1-

5307968 FORWARD LENGTH = 2043

TCATATCC[C/T]CTT

abinit-

GTACTTCTACTACC

gene-

TACAAATATTTTGC

23.322

CAGAAACATATTCA

AGCAG (SEQ ID

NO: 86)

23,679,276
T
G
GGAAGTCGAGAGA
TTT→
F→V
TV
maker-
AT1G53590.1
| Symbols: NTMC2TYPE6.1, NTMC2T6.1 |

TGATGAGTGCTCAA
GTT

N1-

Calcium-dependent lipid-binding (CaLB

CGGTTCCTGAGAAT

augustus-

domain) family protein | chr1:19996315-

GAGTCTGTG[T/G]T

gene-

20000265 FORWARD LENGTH = 3951

TGGTTCAGAATGTC

23.161

AGTTTGAATATTCT

GATGATGATGATAC

TGCACAT (SEQ ID

NO: 87)

23,679,287
A
T
GATGATGAGTGCTC
GAA→
E→D
TV
maker-
AT1G53590.1
| Symbols: NTMC2TYPE6.1, NTMC2T6.1 |

AACGGTTCCTGAGA
GAT

N1-

Calcium-dependent lipid-binding (CaLB

ATGAGTCTGTGTTT

augustus-

domain) family protein | chr1:19996315-

GGTTCAGA[A/T]TG

gene-

20000265 FORWARD LENGTH = 3951

TCAGTTTGAATATT

23.161

CTGATGATGATGAT

ACTGCACATGGAA

GTGTGCA (SEQ ID

NO: 88)

23,679,396
C
A
CAAGGAAAGCAAA
CTT→
L→I
TV
maker-
AT1G53590.1
| Symbols: NTMC2TYPE6.1, NTMC2T6.1 |

AGATGAAGGTAAA
ATT

N1-

Calcium-dependent lipid-binding (CaLB

GGTGTGAGGGCAG

augustus-

domain) family protein | chr1:19996315-

GAGAAGATGGT[C/

gene-

20000265 FORWARD LENGTH = 3951

A]TTGTGAATACAT

23.161

CAGCAAACTCTAAA

GAAGATTCTAGAG

GTGTTACACAT

(SEQ ID NO: 89)

23,886,929
G
A
CGATCCTCGCTACG
TAC→
None
TS
maker-
AT3G14270.1
| Symbols: FAB1B | phosphatidylinositol-

GAACTCTCCACTTC
TAT

N1-

4-phosphate 5-kinase family protein |

CAAAGCTAGTGGA

augustus-

chr3:4753925-4761456 FORWARD

AGGCCGCAA[G/A]T

gene-

LENGTH = 7532

ATCCCCACGCACCT

23.235

GAGGCATCTCCCTC

ATTATCTTCTTCCTC

GTCAAA (SEQ ID

NO: 90)

23,925,895
C
A
AGATAAAAACCGA
CCA→
P→Q
TV
augustus_
AT3G14205.1
| Symbols: | Phosphoinositide

ACCTTTGGAAGGT
CAA

masked-

phosphatase family protein |

TCTCAAGCTAGACA

N1-

chr3:4715707-4720951 REVERSE

GAACAGAGC[C/A]

abinit-

LENGTH = 5245

AACAGAGCTCAACT

gene-

TTCACCAAGACTCA

23.587

ACTCCTTACACAGA

ATCCGAAT (SEQ ID

NO: 91)

23,963,309
CTT
---
CACATTAAACCGGA
GCT,
AS→A
Deletion
augustus_
AT3G14075.2
| Symbols: | Monoidi-acylglycerol lipase,

AGCTAATCGCCGGT
TCT→

(tandem
masked-

N-terminal;Lipase, class 3 | chr3:4663569-

GTCGACGACGAGA
GCT

repeat)
N1-

4667079 REVERSE LENGTH = 3511

GCTCCGAAG[CTT/]

abinit-

CTTCTTCTTCTACTA

gene-

CTCATGGTGCTTCT

23.596

TTGAGGATTGATCG

TGTTTCG (SEQ ID

NO: 92)

24,029,270
G
A
ATTATCTATTGATA
CCT→
P→S
TS
maker-
AT3G13950.1
| Symbols: | unknown protein; BEST

CCTGAGTCTTAAGC
TCT

N1-

Arabidopsis thaliana protein match is:

CGCTGATGACTAGT

augustus-

unknown protein (TAIR:AT4G13266.1);

AGAATGAG[G/A]CT

gene-

Has 339 Blast hits to 265 proteins in 12

CATCGAAGCAGAA

24.76

species: Archae-0; Bacteria-0; Metazoa-

AATAAAACGGTACT

0; Fungi-0; Plants-339; Viruses -0; Other

GCTAATAACCAATC

Eukaryotes-0 (source: NCBI BLink). |

CAAGATA (SEQ ID

chr3:4604149-4605425 FORWARD

NO: 93)

LENGTH = 1277

24,029,279
A
T
TGATACCTGAGTCT
TTC→
F→I
TV
maker-
AT3G13950.1
| Symbols: | unknown protein; BEST

TAAGCCGCTGATGA
ATC

N1-

Arabidopsis thaliana protein match is:

CTAGTAGAATGAG

augustus-

unknown protein (TAIR:AT4G13266.1);

GCTCATCGA[A/T]G

gene-

Has 339 Blast hits to 265 proteins in 12

CAGAAAATAAAAC

24.76

species: Archae-0; Bacteria-0; Metazoa-

GGTACTGCTAATAA

0; Fungi-0; Plants-339; Viruses-0; Other

CCAATCCAAGATAT

Eukaryotes-0 (source: NCBI BLink). |

ATAGAGAC (SEQ ID

chr3:4604149-4605425 FORWARD

NO: 94)

LENGTH = 1277

24,029,294
G
T
AAGCCGCTGATGAC
CGT→
R→S
TV
maker-
AT3G13950.1
| Symbols: | unknown protein; BEST

TAGTAGAATGAGG
AGT

N1-

Arabidopsis thaliana protein match is:

CTCATCGAAGCAGA

augustus-

unknown protein (TAIR:AT4G13266.1);

AAATAAAAC[G/T]G

gene-

Has 339 Blast hits to 265 proteins in 12

TACTGCTAATAACC

24.76

species: Archae-0; Bacteria-0; Metazoa-

AATCCAAGATATAT

0; Fungi-0; Plants-339; Viruses-0; Other

AGAGACCACGCTTG

Eukaryotes-0 (source: NCBI BLink). |

GAACTCT (SEQ ID

chr3:4604149-4605425 FORWARD

NO: 95)

LENGTH = 1277

24,078,887
C
T
CCTCTGTATTTGAC
TCA→
S→L
TS
maker-
AT3G07690.1
| Symbols: | 6-phosphogluconate

ATCTCTTACATCTAT
TTA

N1-

dehydrogenase family protein |

AAATGTCCCTTATT

augustus-

chr3:2457108-2459552 FORWARD

TTTACTT[C/T]ACTG

gene-

LENGTH = 2445

TAAGTTTGGACCCA

24.6

AATCTTGTCTTGTT

GAAAATTCTGCCAT

TGAA (SEQ ID NO:

96)

24,355,223
T
C
GGTTTCGGCCTTTC
None
None
TS
fgenesh_
AT3G13220.1
| Symbols: WBC27, ABCG26 | ABC-2 type

AAAAAAAACTGTTA

masked-

transporter family protein |

ACCAAATTTCGAAA

N1-

chr3:4247968-4250703 REVERSE

TGAACGAA[T/C]CG

abinit-

LENGTH = 2736

AATTAATCAAAATT

gene-

TTGAACTGAAATTA

24.288

CCATAGTTTAATTG

AATTTT (SEQ ID

NO: 97)

24,497,060
A
T
CGCAAACGCCAAGT
TCA→
S→T
TV
maker-
AT3G12660.1
| Symbols: FLA14 | FASCICLIN-like

CAGGAGCATTAATC
ACA

N1-

arabinogalactan protein 14 precursor |

GCTGAAACTCCACT

augustus-

chr3:4019060-4019827 FORWARD

TGTTGTTG[A/T]GT

gene-

LENGTH = 768

TGGCTTTTGCAGCA

24.102

GAAGGTGGCTTAG

CAGTAGCGGCAGT

AGCAGCAG (SEQ ID

NO: 98)

24,542,627
T
A
GAGGTTTCTACACT
TGC→
C→S
TV
maker-
AT3G12500.1
| Symbols: ATHCHIB, PR3, PR-3, CHI-B, B-

TATGATGCCTTTAT
AGC

N1-

CHI, HCHIB | basic chitinase |

CACCGCCGCTAAAT

augustus-

chr3:3962382-3963984 REVERSE

ATTTCCCT[T/A]GCT

gene-

LENGTH = 1603

TCTGCAACAATGGA

24.42

GACACTGCCGCAA

GGAAGAAAGAGCT

CTCTGCC (SEQ ID

NO: 99)

24,717,963
A
T
AATCGCAAATGTTT
ATG→
M→K
TV
fgenesh_
AT3G11980.1
| Symbols: M52, FAR2 | Jojoba acyl CoA

CCTGTCACAGGGAC
AAG

masked-

reductase-related male sterility protein |

AAGCTTGTCTAACA

N1-

chr3:3814236-3817117 FORWARD

TGAAAGAC[A/T]TG

abinit-

LENGTH = 2882

AAAGATGCTCCATG

gene-

AGTCTCTCTTAGAT

24.356

TTTTAAAAAGCTCT

GCATCT (SEQ ID

NO: 100)

24,717,966
A
T
CGCAAATGTTTCCT
TTC→
F→Y
TV
fgenesh_
AT3G11980.1
| Symbols: M52, FAR2 | Jojoba acyl CoA

GTCACAGGGACAA
TAC

masked-

reductase-related male sterility protein |

GCTTGTCTAACATG

N1-

chr3:3814236-3817117 FORWARD

AAAGACATG[A/T]A

abinit-

LENGTH = 2882

AGATGCTCCATGAG

gene-

TCTCTCTTAGATTTT

24.356

TAAAAAGCTCTGCA

TCTAAC (SEQ ID

NO: 101)

TABLE 13

SNPs in chromosome N6 distributed across the QTL3 interval

Ref
Alt

Codon
AA

Affected

Position
nt
nt
Sequence
Change
Change
Type
gene
Ortholog
Description

19,027,546
G
A
TATCTTATTAAAATA
None
None
TS
None
None
None

GAAATACATTTAAA

GAATATTTGGAAAC

ATAAATA[G/A]CAGT

AAAAAAATATAATAT

TATTTGAAAACATAG

ATATCAATATATTAA

A (SEQ ID NO: 102)

19,085,314
C
T
CTTAGACAAAGACTC
GGT→
G→S
TS
maker-
#N/A
#N/A

AGCGGACTCTTTGTG
AGT

N6-

CACAATAGTAACGG

augustus-

CATCAC[C/T]GTCTT

gene-

CAGGCACAGGAAGA

19.60

GACGAAATGGTTTC

AGATTCCCTGGGAG

GAC (SEQ ID NO:

103)

19,156,645
G
T
TTCAAGTCCCTAACC
None
None
TV
maker-
AT5G24740.1
| Symbols: | Protein of unknown function

TCTCTCAAAGCCATA

N6-

(DUF1162) | chr5:8469951-8489703

AAAGCTCTTAACTAG

augustus-

REVERSE LENGTH = 19753

AGAAT[G/T]AATTCA

gene-

AAGACCAATAGTAT

19.65

GTTCTTCATTCCCAC

TATTTATAGCCTATC

(SEQ ID NO: 104)

19,199,109
G
A
GATCATGTTCCGTTT
CGG→
None
TS
maker-
AT5G24830.1
| Symbols: | Tetratricopeptide repeat

TGGATACTTATGAG
CGA

N6-

(TPR)-like superfamily protein |

GAAATACACCAAGT

augustu

chr5:8530978-8533867 FORWARD

TGAGGCG[G/A]CTA

s-gene-

LENGTH = 2890

GACGAGGCGTATTT

19.10

AGCTTACAAAAAAT

GGCTAATGACGAGG

AGCAG (SEQ ID NO:

105)

19,325,186
G
C
GCGTTGAAAAACTTC
GAC→
D→E
TV
maker-
AT5G10930.1
| Symbols: CIPK5, SnRK3.24 | CBL-

GGCGACACCACCAC
GAG

N6-

interacting protein kinase 5 |

CGCCGGTACCGCCG

fgenesh

chr5:3445366-3447114 REVERSE

CTCCGTC[G/C]TCCT

-gene-

LENGTH = 1749

CCTCCTCGCCGTCGT

19.350

TAACGACCGTGATC

ATTGGAGCCTTGAA

GCT (SEQ ID NO:

106)

19,402,086
T
C
TTAAGCTCAAGAAA
TTC→
F→L
TS
augustus_
#N/A
#N/A

GCCAAAGTCAGACC
CTC

masked-

TGTTCTGTTCTCGTG

N6-

TCCGACG[T/C]TCCG

abinit-

GTGGGGAAACCGGC

gene-

AGCCACGGTCGAGA

19.165

AAAGAAGCTGAATC

AGCG (SEQ ID NO:

107)

19,513,420
A
C
TATATATAGGTGGAT
ATT→
I→L
TV
maker-
AT5G25430.1
| Symbols: | HCO3-transporter family |

CTCTGAGTGCTGTG
CTT

N6-

chr5:8851251-8854259 FORWARD

GAAACATTAGCTTCT

augustus-

LENGTH = 3009

ACTTCT[A/C]TTTGC

gene-

GGAATCATCCACGC

19.27

CATCTTTGGTGGACA

GCCATTGTTGATACT

T (SEQ ID NO: 108)

19,583,431
T
C
TGTTTTAATAGGACG
None
None
TS
None
None
None

AGCTATGGGAAGCC

TGTTTAACAATGATG

GGCCTG[T/C]CTGCA

AACCCTGAAACGTCT

CCCCAGAATTTAGCA

TTGTACAAACTTTAA

(SEQ ID NO: 109)

19,601,021
G
A
TCCGAAATACCCAAA
None
None
TS
None
None
None

AATACCTAATCTAAA

CAAAATATTTCGCAT

ACTTT[G/A]GGTACC

CGATCGGGTCTCCG

GTAAGATCCAGACC

CAAACCGAGATCGT

AT (SEQ ID NO: 110)

19,706,563
C
T
TACATGCTAACAGTG
GAG→
None
TS
maker-
AT5G259
| Symbols: | Protein of Unknown Function

ATGTGATATTGTTCA
GAA

N6-
50.1
(DUF239) | chr5:9057793-9059963

CTCGAGGGAGTTGA

augustus-

REVERSE LENGTH = 2171

AACGGG[C/T]TCTAC

gene-

GGCTGCACCCAGGG

19.94

CGAATCTAGTTGTTG

TTTGGACAAAACCG

GA (SEQ ID NO: 111)

19,800,643
C
T
GATGCTTCCACCGAC
GGC→
None
TS
augustus_
AT5G40890.1
| Symbols: ATCLC-A, CLC-A, CLCA, ATCLCA

GGCGTTGGCTTACTC
GGT

masked-

| chloride channel A | chr5:16381346-

AGCTCCCCCCGTGAC

N6-

16385319 REVERSE LENGTH = 3974

GGCGG[C/T]GGTGG

abinit-

CGTTGATAGTCTTGA

gene-

TTACGAGGTTATCGA

19.239

GAATTACGCTTACAG

(SEQ ID NO: 112)

19,906,666
A
C
CCGTGTGGTGGTAG
GAA→
E→D
TV
maker-
#N/A
#N/A

AATCATCGACGAGG
GAC

N6-

TTTGCGTCAAGGAG

augustus-

GAGTACGA[A/C]ACT

gene-

CGTCCCGGGAAGCG

19.53

CTTCTTCAGCTGCAT

AAACTACGAGGTAA

CCCA (SEQ ID NO:

113)

20,000,119
A
G
TATAAAGCCAGCGG
TAC→
Y→C
TS
maker-
AT5G26810.1
| Symbols: | Pectin lyase-like superfamily

TAGCGCTTACGGTTT
TGC

N6-

protein | chr5:9430952-9432969

ATGGCGATAAATCA

fgenesh-

FORWARD LENGTH = 2018

GCGTTCT[A/G]CAAC

gene-

TGCGGTTTCTTGGG

20.335

GTTACAAGATACGTT

GTGGGATGTTCAAG

GCA (SEQ ID NO:

114)

20,095,002
C
T
AGGTCTTCTGCCAAA
GTG→
V→M
TS
maker-
AT2G01390.1
| Symbols: | Tetratricopeptide repeat

CCCAAGCCTGTGGA
ATG

N6-

(TPR)-like superfamily protein |

TCAACGTTGCTCCTT

augustus-

chr2:172256-174137 FORWARD

GGTACA[C/T]ACCAA

gene-

LENGTH = 1882

GTGAGTGATGGGCT

20.62

TTCACCATCTCCTTTA

CTGCTTCGATAAGTT

(SEQ ID NO: 115)

20,205,211
G
A
CAATAATGTGAACTC
CTG→
None
TS
augustus_
AT5G27470.1
| Symbols: | seryl-tRNA synthetase/

TCTTTTCTTCAAGAA
TTG

masked-

serine--tRNA ligase | chr5:9695008-

GTCTAGCCCAAACCT

N6-

9697389 FORWARD LENGTH = 2382

GATCA[G/A]AGCCT

abinit-

GGTTAAGTAGCACA

gene-

CCATCTCCTTTAAGG

20.166

AAAAAGCCTCTTCCT

C (SEQ ID NO: 116)

20,300,571
G
A
CGTCGATAGTTCACA
CGC→
None
TS
maker-
AT5G27320.1
| Symbols: ATGID1C, GID1C | alpha/beta-

GAGACAACAACGAA
CGT

N6-

Hydrolases superfamily protein |

ACCGCACAGACCAA

fgenesh-

chr5:9629087-9631210 FORWARD

CAAGCCT[G/A]CGG

gene-

LENGTH = 2124

CAAAGAGTGTCATA

20.419

GATAGCACTGTTTGC

AGAAGAGTGCACAA

AGCT (SEQ ID NO:

117)

20,406,148
C
T
GTTACAGATACGAG
CTA→
None
TS
augustus_
AT5G28210.1
| Symbols: | mRNA capping enzyme family

ATGGAGCCGTTTGG
TTA

masked-

protein | chr5:10188586-10190463

TGTACGGTTAAAGG

N6-

FORWARD LENGTH = 1878

CCTTTTGC[C/T]TACT

abinit-

CTCTTCCGTGGAGAA

gene-

GAAGGTGTTCAACG

20.212

AGCTGATACCTTCGC

TC (SEQ ID NO: 118)

20,407,023
G
T
TAATCCCTTAGATTC
None
None
TV
None
None
None

AAGCAGCAACGCCT

GTAGGCTCCCTGAG

ATTCATT[G/T]CCCA

TTATGTGTCTTAGCC

ATCAATCTAATTGCA

TAAACTAGTAAGAA

CA (SEQ ID NO: 119)

20,505,840
A
G
GCCGGTCTAGCACA
GAT→
None
TS
maker-
AT5G28680.1
| Symbols: ANX2 | Malectin/receptor-like

TAAGATCTCAAACAA
GAC

N6-

protein kinase family protein |

AACAACTCCAAAAG

augustus-

chr5:10719437-10722013 REVERSE

AGTACAC[A/G]TCTG

gene-

LENGTH = 2577

ACTTCTCGGTTAACT

20.93

GCTGTCTCCTAAAGT

ACTCTGGATCTAAGT

A (SEQ ID NO: 120)

20,601,198
G
A
TGGTTGGACTGTGC
ACG→
None
TS
fgenesh_
AT5G49580.1
| Symbols: | Chaperone DnaJ-domain

AATCCGAGGCTTTG
ACA

masked-

superfamily protein | chr5:20123823-

ACTCTTTCATCAGGA

N6-

20126813 REVERSE LENGTH = 2991

TGGGGAC[G/A]GCA

abinit-

TCGTTTTTCTCAATC

gene-

ATGTGGTGTGGTGT

20.524

CTTCTCAGCGTTTTC

CAT (SEQ ID NO:

121)

20,631,917
T
C
CTTGGATAAGAGTTT
None
None
TS
None
None
None

ATAGGAAAACCAAA

ATTCGCTTGCAATGA

GTATCA[T/C]TATTA

AACCGATCATGAGG

AGATCATAACAACTA

GAATTTAAATGATGT

T (SEQ ID NO: 122)

20,702,631
T
C
TTGTGTAGATGGTC
AAT→
None
TS
maker-
AT5G49330.1
| Symbols: MYB111, ATMYB111, PFG3 |

GCATATTGCAACACA
AAC

N6-

myb domain protein 111 |

TCTACCAGGAAGAA

augustus-

chr5:19998952-20001378 REVERSE

CAGACAA[T/C]GAA

gene-

LENGTH = 2427

ATTAAAAACTATTGG

20.37

AATTCACATCTCAGC

CGCAAAATCTATGCC

TT (SEQ ID NO: 123)

20,805,841
G
A
ACAGGGTATACCTG
CTC→
None
TS
maker-
AT3G06930.2
| Symbols: ATPRMT4B, PRMT4B | protein

TGGCTGAGACATTCT
CTT

N6-

arginine methyltransferase 4B |

ATAATAAGGCTCCTT

augustus-

chr3:2185143-2189387 REVERSE

CAAGTC[G/A]AGTTT

gene-

LENGTH = 4245

GCACGATGATGTTT

20.112

GGAGGATTCCACCTT

GATTGGCACCGGGC

CC (SEQ ID NO: 124)

20,816,431
C
A
CTCCCTACTGATTTC
None
None
TV
None
None
None

TCCAGGTTTAAGAC

GATGCACATGTACG

ATATCGT[C/A]GTCA

AGAACCGCAACATG

TTCCAAGTAAGGGA

TATGTAGCCATATTT

TGG (SEQ ID NO:

125)

20,846,412
C
A
AAACGAGATGATTTT
None
None
TV
None
None
None

CCTTTAAACGTTTAA

AAAAAATCAATCTA

GGCATT[C/A]AAAAA

ATCGGTCTGCCAATA

CTATTAGCTATTTTCT

GAACTTTGGTTGCT

(SEQ ID NO: 126)

20,902,530
G
A
GCAGCGGTTCGCTC
TCC→
None
TS
augustus_
AT5G48890.1
| Symbols: | C2H2-like zinc finger protein

CTTCTTGTGGGCGTT
TCT

masked-

| chr5:19820353-19820874 FORWARD

CTGGTGGCCTCCTAA

N6-

LENGTH = 522

GGCTTG[G/A]GAAC

abinit-

TCTGGAACTTTCTAG

gene-

AACAAAAGAGGCAA

20.311

GAGAATACTCTTGAT

GA (SEQ ID NO: 127)

21,001,046
G
A
TTTAGGAAGATTAG
AAC→
None
TS
maker-
#N/A
#N/A

ACAGAACATACCACT
AAT

N6-

GTCAGTAGCTTTGAC

fgenesh-

GATGGC[G/A]TTACT

gene-

GATAAGAGGCACTT

21.260

TAGAACACATGGCA

GCAACTACCTGCATG

CT (SEQ ID NO: 128)

21,100,769
G
A
TCCCTCTGTCAAAAC
TCG→
None
TS
maker-
AT5G48250.1
| Symbols: | B-box type zinc finger protein

TGTGACTGGTTAGG
TCA

N6-

with CCT domain | chr5:19561319-

CCACAACGGAGCTA

fgenesh-

19563722 REVERSE LENGTH = 2404

CTAATTC[G/A]CATC

gene-

ATAAGAAGCAAACC

21.198

ATTAACTGCTACTCT

GGTTGCCCCTCGAGT

GA (SEQ ID NO: 129)

21,199,766
G
A
CTGTGATAAACGATC
CTG→
None
TS
augustus_
AT5G47840.1
| Symbols: AMK2 | adenosine

TTTGACCATCATTAC
TTG

masked-

monophosphate kinase | chr5:19375441-

AACTATTTCATTAGG

N6-

19378339 FORWARD LENGTH = 2899

GACCA[G/A]TTGTCC

abinit-

TTTCTCCATGTGTTCT

gene-

TTTGCCAGTCTTCCA

21.377

TTCTCACTCCCAG

(SEQ ID NO: 130)

21,218,604
G
T
GGAACTATTTTCTAA
None
None
TV
None
None
None

GTGGATCAACTCAA

ACGACGCCGTTTCGC

TTCTAT[G/T]TAAGT

CAAAAGTCCTCTCTT

TTATGTTTTGTATCC

AAAGTATACAGCTTT

(SEQ ID NO: 131)

TABLE 14

SNPs in chromosome N6 that are in candidate genes within the QTL3 interval

Ref
Alt

Codon
AA

Affected

Position
nt
nt
Sequence
Change
Change
Type
gene
Ortholog
Description

19,336,744
A
G
GAGTTTTGCAGCAGCAGATTACT
AAG→
K→R
TS
maker-N6-
AT5G25120.1
| Symbols: CYP71611 | ytochrome p450,

TCCCGGTAATTGGTAAATTAATCG
AGG

fgenesh-

family 71, subfamily B, polypeptide 11 |

ATA[A/G]GATCACAGGGTTACAT

gene-

chr5:8662099-8664533 FORWARD

AGCAAATGTGAGAAGGTTTTTAA

19.294

LENGTH = 2435

AGCAATGGATG (SEQ ID NO:

132)

19,336,819
G
A
TGAGAAGGTTTTTAAAGCAATGG
CGT→
R→H
TS
maker-N6-
AT5G25120.1
| Symbols: CYP71611 | ytochrome p450,

ATGCATTTTTCGATCAATCTATAA
CAT

fgenesh-

family 71, subfamily B, polypeptide 11 |

AGC[G/A]TCATCTTGAAGACGAG

gene-

chr5:8662099-8664533 FORWARD

AGCCTTGAAGATGATATCATAGC

19.294

LENGTH = 2435

CTTGCTCTTAA (SEQ ID NO: 133)

19,337,615
A
C
TTGATCTTGAAGAATCATATGGA
AAG→
K→Q
TV
maker-N6-
AT5G25120.1
| Symbols: CYP71611 | ytochrome p450,

CTCGTATGTCCTAAGAAAGTTCCA
CAG

fgenesh-

family 71, subfamily B, polypeptide 12 |

CTT[A/C]AGCTTATCCCGATTCTTA

gene-

chr5:8662099-8664533 FORWARD

CTCAATGGACTTGACTTCGATTTA

19.294

LENGTH = 2435

TATTTTCG (SEQ ID NO: 134)

19,350,156
A
C
ACTGAAACTTCCTATAATTGGCAA
CAT→
H→P
TV
maker-N6-
AT5G25130.1
| Symbols: CYP71612 | cytochrome P450,

CTTGCACCAATTAGGCTCGCAGC
CCT

fgenesh-

family 71, subfamily B, polypeptide 12 |

CTC[A/C]TCGTTCATTAACAAAAT

gene-

chr5:8668299-8670194 FORWARD

TATCTGAAAAGTATGGACCTCTA

19.295

LENGTH = 1896

ATGTCCCTAA (SEQ ID NO: 135)

19,353,584
C
T
GTTGTATGAAAGTCTTGCAGCGT
GAT→
D→N
TS
maker-N6-
AT5G25140.1
| Symbols: CYP71613 | cytochrome P450,

ACGTCAAGTAAGGTCGTGAACAA
AAT

augustus-

family 71, subfamily B, polypeptide 13 |

CAAT[C/T]AACGTCGAACGTTTTC

gene-

chr5:8672424-8674629 FORWARD

AAGACATCTTTCACTGTCTCTGGA

19.75

LENGTH = 2206

GTTGACGCCA (SEQ ID NO: 136)

19,353,648
A
C
TTCAAGACATCTTTCACTGTCTCT
AAT→
N→K
TV
maker-N6-
AT5G25140.1
| Symbols: CYP71613 | cytochrome P450,

GGAGTTGACGCCACAACGGTAGA
AAG

augustus-

family 71, subfamily B, polypeptide 13 |

CAC[A/C]TTTCCAAGCTTGAGGGA

gene-

chr5:8672424-8674629 FORWARD

CATTAGAGCTCCATATTTTTCAGA

19.75

LENGTH = 2206

TAATTTGAA (SEQ ID NO: 137)

19,353,749
G
T
CATGGAACGATGAGGTTTGGATC
CTT→
L→I
TV
maker-N6-
AT5G25140.1
| Symbols: CYP71613 | cytochrome

CCAGTTGGTGCAAGTTACCAATT
ATT

augustus-

family 71, subfamily B, polypeptide

ATAA[G/T]AAGCCTTGGTGGTCC

gene-

chr5:8672424-8674629 FORWARD

AGGAGGTAGGTTTTTCTTTGTCTT

19.75

LENGTH = 2206

TCTCGTGTTCT (SEQ ID NO: 138)

19,476,836
A
C
CTACATATGCATAGTTTCCATACA
None
None
TV
maker-N6-
AT1G52570.1
| Symbols: PLDALPHA2 | phospholi

GAGAGATAGATAGCAGTAGGTAT

augustus-

alpha 2 | chr1:19583940-19587050

AAG[A/C]TAAGCTAAGCTAACCT

gene-

REVERSE LENGTH = 3111

ACAGATATAATGAAACAATTACA

19.80

AGATGTAAAAA (SEQ ID NO:

ACAGATATAATGAAACAATTACA

AGATGTAAAAA (SEQ ID NO:

139)

19,783,834
G
C
TTTAGCTGTGTTCCTCACCTCATTC
CAG→
Q→E
TV
augustus_
AT5G26220.1
| Symbols: | ChaC-like family protein |

GCCAATTCTATCACGAACTCTTCC
GAG

masked-N6-

chr5:9162969-9164685 REVERSE

T[G/C]ATGCTCTGTTTTGACAACA

abinit-

LENGTH = 1717

TTCATCAAACAAATCAACGCCAAA

gene-

AATCTTA (SEQ ID NO: 140)

19.236

19,784,007
T
C
TTGAAAAGGTACTCCCGGTTGTTC
AAA→
K→R
TS
augustus_
AT5G26220.1
| Symbols: | ChaC-like family protein |

CCACAAGGTTCAGAAGCTGTGGC
AGA

masked-N6-

chr5:9162969-9164685 REVERSE

GAT[T/C]TGCATGGCCATCTCCTC

abinit-

LENGTH = 1717

CAAAAGTAGTATTTGTTCGACACT

gene-

TTGTCTGGT (SEQ ID NO: 141)

19.236

19,784,367
T
C
TCGACAAGGGTTTTAGAGTCGTA
ATA→
I→M
TS
augustus_
AT5G26220.1
| Symbols: | ChaC-like family protein |

TTCACACTCTCTTCGTTCCAAGTA
ATG

masked-N6-

chr5:9162969-9164685 REVERSE

CTC[T/C]ATGGCTAGCTTCTCCTTC

abinit-

LENGTH = 1717

TCAGGTCCTCCATGAACACAATAA

gene-

GCAGCACC (SEQ ID NO: 142)

19.236

19,784,633
A
G
CAACATTGATCTATTTCTTTGTTG
TTT→
F→L
TS
augustus_
AT5G26220.1
| Symbols: | ChaC-like family protein |

ATAATTCAAGAACAAGTGTGTTG
CTT

masked-N6-

chr5:9162969-9164685 REVERSE

TAA[A/G]CATACCGAGATCAAAG

abinit-

LENGTH = 1717

ACGCGTTTGTAGTCTTTAATATTG

gene-

CCAATGAGTT (SEQ ID NO: 143)

19.236

19,784,672
T
A
GTGTGTTGTAAACATACCGAGAT
AAT→
N→Y
TV
augustus_
AT5G26220.1
| Symbols: | ChaC-like family protein |

CAAAGACGCGTTTGTAGTCTTTAA
TAT

masked-N6-

chr5:9162969-9164685 REVERSE

TAT[T/A]GCCAATGAGTTTTTCGT

abinit-

LENGTH = 1717

CAAAATCGAAACCTGGGTTCCAT

gene-

ATTATAGATC (SEQ ID NO: 144)

19.236

19,784,688
G
T
CCGAGATCAAAGACGCGTTTGTA
GAC→
D→E
TV
augustus_
AT5G26220.1
| Symbols: | ChaC-like family protein |

GTCTTTAATATTGCCAATGAGTTT
GAA

masked-N6-

chr5:9162969-9164685 REVERSE

TTC[G/T]TCAAAATCGAAACCTGG

abinit-

LENGTH = 1717

GTTCCATATTATAGATCCATATCC

gene-

GAATTCCCA (SEQ ID NO: 145)

19.236

19,784,733
TT
CA
TTTTCGTCAAAATCGAAACCTGG
GAA→
E→V
Subst-
augustus_
AT5G26220.1
| Symbols: | ChaC-like family protein |

GTTCCATATTATAGATCCATATCC
GTG

masked-N6-

chr5:9162969-9164685 REVERSE

GAA[TT/CA]CCCACAATACCATCT

abinit-

LENGTH = 1717

TGAACGTTAAAGTCTCTTTTAGGA

gene-

AGAAGAAATAG (SEQ ID NO:

19.236

146)

19,800,525
A
T
CCTAAATCAAGCAAGCTTCTCGAT
CAC→
H→L
TV
augustus_
AT5G40890.1
| Symbols: ATCLC-A, CLC-A, CLCA, ATCLCA

TCGATTCGACTCGATCATCATCAT
CTC

masked-N6-

| chloride channel A | chr5:16381346-

GC[A/T]CTCGAATCATCTACAGAA

abinit-

16385319 REVERSE LENGTH = 3974

CGGGATCGAATCCGATAATCTCC

gene-

TCTGGTCTC (SEQ ID NO: 147)

19.236

20,191,826
G
A
ACTTAATTCACTTTTTAAAACTGTT
ATC→
None
TS
augustus_
AT5G27600.1
| Symbols: LACS7, ATLACS7 | long-chain

ACTATTATCACACACTACCATTTA
ATT

masked-N6-

acyl-CoA synthetase 7 | chr5:9742576-

A[G/A]ATTGCCTTCTTCTTTGAGT

abinit-

9747005 FORWARD LENGTH = 4430

TATAGGCAAGTTGGAAAAGCCTT

gene-

CTTTTTGTA (SEQ ID NO: 148)

20.163

20,300,548
A
G
AAGGGTAACGATTCTCCGGCGCA
TTC→
F→S
TS
maker-N6-
AT5G27320.1
| Symbols: ATGID1C, GID1C | alpha

CGTCGATAGTTCACAGAGACAAC
TCC

fgenesh-

Hydrolases superfamily protein |

AACG[A/G]AACCGCACAGACCAA

gene-

chr5:9629087-9631210 FORWARD

CAAGCCTGCGGCAAAGAGTGTCA

20.419

LENGTH = 2124

TAGATAGCACTG (SEQ ID NO:

149)

20,375,643
T
A
GATTCTAGCATTTTACTCACAGTG
GAA→
E→D
TV
maker-N6-
AT5G27980.1
| Symbols: | Seed maturation protein |

AGAACATCGGCAAGCGTAGTCTT
GAT

augustus-

chr5:10015774-10016726 REVERSE

TTC[T/A]TCATCGGAGTTAGCTCG

gene-

LENGTH = 953

AGCGTTAAGAGTGGCAGCTGACT

20.83

GAGCAGAAGC (SEQ ID NO: 150)

20,766,637
G
A
TTCTGCCTCTTGAGAGAAAATCAT
CGT→
R→H
TS
maker-N6-
AT4G02280.1
| Symbols: SUS3, ATSUS3 | sucrose

GCCAACGGGTAGGTTCGAGACG
CAT

augustus-

synthase 3 | chr4:994927-998967

ATGC[G/A]TGAATGGGTTCACGA

gene-

FORWARD LENGTH = 4041

CGCCATCTCTGCTCAACGCAATGA

20.39

GCTCCTCTCTC (SEQ ID NO: 151)

20,769,461
A
C
TATAGTCTCTCCAGGAGCTGATAT
AAGV
K→T
TV
maker-N6-
AT4G02280.1
|Symbols: SUS3, ATSUS3 | sucrose

GACCATATACTTTCCTTATTCTGA
ACG

augustus-

synthase 3 | chr4:994927-998967

CA[A/C]GGAAAGAAGACTAACTG

gene-

FORWARD LENGTH = 4041

CCCTTCATGAGTCTATTGAAGAAC

20.39

TTCTGTTTA (SEQ ID NO: 152)

20,770,769
A
G
ATGATAATTTTTTTCTGGTTTTGC
AAT→
N→D
TS
maker-N6-
AT4G02280.1
|Symbols: SUS3, ATSUS3 | sucrose

AGGCCAATTCAATCCCACTGGCA
GAT

augustus-

synthase 3 | chr4:994927-998967

ACT[A/G]ATGAGCATTGAGCAAG

gene-

FORWARD LENGTH = 4041

CTATGGTTGGATTCTAATACTTGC

20.39

TGCACTCCCT (SEQ ID NO: 153)

20,823,998
CGC
TG
TACAAATTTGGCTCAAGTCTGGG
GCG→
AVT
Substi-
maker-N6-
AT5G48960.1
| Symbols: | HAD-superfamily hydrolase,

TCACAAACAACAGCTAGTTTTCCAT
ACA

tution
augustus-

subfamily IG, 5′nucleotidase |

AGC[CGC/TGT]ATCAGGAAGGGG

gene-

chr5:19849543-19853564 FORWARD

AGAATCATGAGCCAGTGACTGCA

20.116

LENGTH = 4022

GAAGAGAGATACAAC (SEQ ID

NO: 154)

20,825,959
T
C
ATGAAATTTAGGTCCTGTGAAGC
AAT→
N→D
TS
maker-N6-
AT5G48960.1
| Symbols: | HAD-superfamily hydrolase,

AAATGACCTTGTACAGTCCTTTGT
GAT

augustus-

subfamily IG, 5′-nucleotidase |

AAT[T/C]AAGAGTGCCAAGATCT

gene-

chr5:19849543-19853564 FORWARD

GCTGAAATAGATCCATCATCCAAT

20.116

LENGTH = 4022

CTATCAACCA (SEQ ID NO: 155)

20,826,301
G
C
TGAATGGTGTTGAAAAGTTTACC
AAC→
N→K
TV
maker-N6-
AT5G48960.1
| Symbols: | HAD-superfamily hydrolase,

ATACCTGACAGCTTTATTTGATAA
AAG

augustus-

subfamily IG, 5′-nucleotidase |

CAT[G/C]TTCGTACCGTGCATGGC

gene-

chr5:19849543-19853564 FORWARD

TCTCTGCACATACCCAAATCTATC

20.116

LENGTH = 4022

AGCCTTGAC (SEQ ID NO: 156)

20,827,570
A
G
TACCGGAGAAGAATAAGCGGTG
TGT→
C→R
TS
maker-N6-
AT5G48960.1
| Symbols: | HAD-superfamily hydrolase,

AACCTCAGCTTCTTCGCCATATCT
CGT

augustus-

subfamily IG, 5′-nucleotidase |

CCAC[A/G]CATAAGCATTCTGTCG

gene-

chr5:19849543-19853564 FORWARD

AACACCTGATGGGCGCATAGATT

20.116

LENGTH = 4022

CATTTTTCACT (SEQ ID NO: 157)

20,827,573
T
G
CGGAGAAGAATAAGCGGTGAAC
ATG→
M→L
TV
maker-N6-
AT5G48960.1
| Symbols: | HAD-superfamily hydrolase,

CTCAGCTTCTTCGCCATATCTCCA
CTG

augustus-

subfamily IG, 5′-nucleotidase |

CACA[T/G]AAGCATTCTGTCGAAC

gene-

chr5:19849543-19853564 FORWARD

ACCTGATGGGCGCATAGATTCAT

20.116

LENGTH = 4022

TTTTCACTTTT (SEQ ID NO: 158)

20,912,356
A
C
AATGTTGTGGCAGGCGTTGTCTA
GAT→
D→A
TV
maker-N6-
AT5G48840.1
| Symbols: PANC, PTS, ATPTS | homolog of

TAAGTAGGTCACTGGCCATGGCT
GCT

fgenesh-

bacterial PANC | chr5:19803557-1

AAAG[A/C]TTCTGCACAACAAGG

gene-

REVERSE LENGTH = 1521

GCAAACCAGTTGTAAAGAGCTTA

20.387

AGGATATGATAA (SEQ ID NO:

159)

20,912,364
C
G
GGCAGGCGTTGTCTATAAGTAGG
CAA→
Q→E
TV
maker-N6-
AT5G48840.1
| Symbols: PANC, PTS, ATPTS | homolog of

TCACTGGCCATGGCTAAAGATTCT
GAA

fgenesh-

bacterial PANC | chr5:19803557-19805077

GCA[C/G]AACAAGGGCAAACCAG

gene-

REVERSE LENGTH = 1521

TTGTAAAGAGCTTAAGGATATGA

20.387

TAATTTCAGAG (SEQ ID NO: 160)

20,912,385
A
G
GGTCACTGGCCATGGCTAAAGAT
AAA→
K→E
TS
maker-N6-
AT5G48840.1
| Symbols: PANC, PTS, ATPTS | homolog of

TCTGCACAACAAGGGCAAACCAG
GAA

fgenesh-

bacterial PANC I chr5:19803557-19805077

TTGT[A/G]AAGAGCTTAAGGATA

gene-

REVERSE LENGTH = 1521

TGATAATTTCAGAGATTGTTGGA

20.387

GCTGCAGGAAGA (SEQ ID NO:

161)

20,912,629
C
G
GTGTGTACAACATGCAGATTGTT
CAG→
Q→E
TV
maker-N6-
AT5G48840.1
| Symbols: PANC, PTS, ATPTS | homolog of

GATCAAGAAACTCTTGAAGGAGT
GAG

fgenesh-

bacterial PANC | chr5:19803557-19805077

AGAA[C/G]AGATAGAGAGTGGA

gene-

REVERSE LENGTH = 1521

GTAGTGATTTGTGTTGCTGCCTG

20.387

GTTTGGAACGGTT (SEQ ID NO:

162)

20,912,710
GCA
AC
CTGCCTGGTTTGGAACGGTTAGG
GCA→
A→T
Substi-
maker-N6-
AT5G48840.1
| Symbols: PANC, PTS, ATPTS | homolog of

TCTCATAGACAACATTGAGATCAAT
ACT

tution
fgenesh-

bacterial PANC | chr5:19803557-19805077

GTC[GCA/ACT]GTCTAATTCCCAC

gene-

REVERSE LENGTH = 1521

TTTCCGCATCCATCTCTGGTTCGT

20.387

TGACAGGAGCTTC (SEQ ID NO:

163)

20,926,866
C
G
AAAATCCGAGCTCTTGTTCTGGTT
GCT→
A→P
TV
augustus_
AT5G48810.1
| Symbols: ATB5-6, B5 #3, ATCB5-D, CB5-D

AGACTGGGTCTGGTTTGAAGGAG
CCT

masked-N6-

| cytochrome B5 isoform D |

GAG[C/G]AACAAACTTGGTTTTG

abinit-

chr5:19789067-19790334 REVERSE

GCTGGGACAGTGGCGGAGTCAA

gene-

LENGTH = 1268

TATCACCGACGT (SEQ ID NO:

20.316

164)

20,926,884
C
T
CTGGTTAGACTGGGTCTGGTTTG
GCC→
A→T
TS
augustus_
AT5G48810.1
| Symbols: ATB5-6, B5 #3, ATCB5-D, CB5-D

AAGGAGGAGCAACAAACTTGGTT
ACC

masked-N6-

| cytochrome B5 isoform D |

TTGG[C/T]TGGGACAGTGGCGGA

abinit-

chr5:19789067-19790334 REVERSE

GTCAATATCACCGACGTAGTACTC

gene-

LENGTH = 1268

ATCAAGCATGG (SEQ ID NO:

20.316

165)

20,927,474
T
G
CGTCGATGACGATCCAACAGTCC
GAG→
→A
TV
augustus_
AT5G48810.1
| Symbols: ATB5-6, B5 #3, ATCB5-D, CB5-D

TGGTTGCTAGTGTGCTGAGAAAC
GCG

masked-N6-

| cytochrome B5 isoform D |

CTCC[T/G]CCAAGGTGAACACTTT

abinit-

chr5:19789067-19790334 REVERSE

GCCGTCTCCGCCCATCTCCCGATG

gene-

LENGTH = 1268

ATCCTCAATT (SEQ ID NO: 166)

20.316

21,136,893
G
C
AGCGTTCGGATTCTTTACGAACAT
CGA→
R→P
TV
maker-N6-
AT5G48100.1
| Symbols: TT10, LAC15, ATLAC15 |

CAAAAGCTTATACTCCGGACAAG
CCA

augustus-

Laccase/Diphenol oxidase family protein |

TTC[G/C]AGTCAAAATCTCACGTA

gene-

chr5:19489327-19492744 REVERSE

GAATAATCTCAACGGTTTCACTAA

21.546

LENGTH = 3418

ATCTTCTAG (SEQ ID NO: 167)

21,137,125
T
G
GGAACGCGGTTTCCTGCGTTTCC
GAT→
D→E
TV
maker-N6-
AT5G48100.1
| Symbols: TT10, LAC15, ATLAC15 |

GCCTCTGGTTTTCAATTTTACCGC
GAG

augustus-

Laccase/Diphenol oxidase family protein |

GGA[T/G]GATCAACCGTTGATTTT

gene-

chr5:19489327-19492744 REVERSE

GCAGACTTCGAGACTTGCTACGG

21.546

LENGTH = 3418

AAGTAAAGAT (SEQ ID NO: 168)

21,137,150
T
C
CTCTGGTTTTCAATTTTACCGCGG
TCG→
S→P
TS
maker-N6-
AT5G48100.1
| Symbols: TT10, LAC15, ATLAC15 |

ATGATCAACCGTTGATTTTGCAGA
CCG

maker-N6-

Laccase/Diphenol oxidase family protein |

CT[T/C]CGAGACTTGCTACGGAA

augustus-

chr5:19489327-19492744 REVERSE

GTAAAGATGATTAAGTACGGAGA

gene-

LENGTH = 3418

AGCGGTTGAA (SEQ ID NO: 169)

21,137,202
C
T
GAGACTTGCTACGGAAGTAAAGA
ACG→
T→M
TS
maker-N6-
AT5G48100.1
| Symbols: TT10, LAC15, ATLAC15 |

TGATTAAGTACGGAGAAGCGGTT
ATG

augustus-

Laccase/Diphenol oxidase family protein |

GAAA[C/T]GGTTTTTCAAGGGAC

gene-

chr5:19489327-19492744 REVERSE

GAGTTTAGGTGGTGGTGGAATCG

21.546

LENGTH = 3418

ACCACCCCATGC (SEQ ID NO:

170)

21,194,862
G
A
ACTATGATCTCAGAAGTTTTGGAC
ACC→
T→I
TS
maker-N6-
AT5G47860.1
| Symbols: | Protein of unknown function

CCATATTTGAAGCCATCCAAGAT
ATC

augustus--

(DUF1350) | chr5:19381519-19384422

GTG[G/A]TTGCATCGACGAGCTG

gene

FORWARD LENGTH = 2904

GGCAAGATCTTTGGACGTGTATG

21.614

CACGAAGAATC (SEQ ID NO:

171)

21,194,879
GGC
AGT
TTTGGACCCATATTTGAAGCCATC
GCC→
A→T
Substi-
maker-N6-
AT5G47860.1
|Symbols: | Protein of unknown function

CAAGATGTGGTTGCATCGACGAG
ACT

tution
augustus--

(DUF1350) | chr5:19381519-19384422

CTG[GGC/AGT]AAGATCTTTGGA

gene

FORWARD LENGTH = 2904

CGTGTATGCACGAAGAATCTTCG

21.614

AGTCCATTCCAAGCG (SEQ ID

NO: 172)

21,194,895
G
A
AAGCCATCCAAGATGTGGTTGCA
ACG→
T→M
TS
maker-N6-
AT5G47860.1
| Symbols: | Protein of unknown function

TCGACGAGCTGGGCAAGATCTTT
ATG

augustus--

(DUF1350) | chr5:19381519-19384422

GGAC[G/A]TGTATGCACGAAGAA

gene

FORWARD LENGTH = 2904

TCTTCGAGTCCATTCCAAGCGTAC

21.614

CTCCAACCTCT (SEQ ID NO: 173)

21,194,899
A
C
CATCCAAGATGTGGTTGCATCGA
TAC→
Y→D
TV
maker-N6-
AT5G47860.1
| Symbols: | Protein of unknown function

CGAGCTGGGCAAGATCTTTGGAC
GAC

augustus--

(DUF1350) | chr5:19381519-19384422

GTGT[A/C]TGCACGAAGAATCTTC

gene

FORWARD LENGTH = 2904

GAGTCCATTCCAAGCGTACCTCCA

21.614

ACCTCTTTAG (SEQ ID NO: 174)

21,196,738
G
C
ACTCCATCCAATACAGCTAAGCTA
CTC→
L→V
TV
maker-N6-
AT5G478
|Symbols: | Protein of unknown function

CTGCGAAAGAAAATCAATCACCT
GTC

augustus--
60.1
(DUF1350) | chr5:19381519-19384422

CGA[G/C]TTCTGACCAGTCGGCG

gene

FORWARD LENGTH = 2904

GATCCAGCGGAGCTACTTTGAGA

21.614

TTGCTTGACAG (SEQ ID NO: 175)

21,197,016
A
G
GACTCTTCGTGAGACGCGCTCTTT
TCT→
S→P
TS
maker-N6-
AT5G47860.1
|Symbols: | Protein of unknown function

GTCGAATCTGATCACCGAGAATG
CCT

augustus--

(DUF1350) | chr5:19381519-19384422

GAG[A/G]CCGGCGAGTGGAAGA

gene

FORWARD LENGTH = 2904

GAAGTTAGAGAGTCTCCGGCGG

21.614

GGAGAGAAAGACG (SEQ ID NO:

176)

It can be hypothesized that some of these genes which contain SNPs in genes which have or are predicted to have a function in lipid biosynthesis or a related pathway in the parent which increases PUFA could be contributing to the increase in PUFA by some functional difference in those genes. For example, a desaturase enzyme may prefer different substrates depending on minor changes in its substrate binding pocket, which could impact the amount of PUFA produced.

Example 2: Crosses/Hybrids

As demonstrated herein, adding certain genetic elements (QTL) can increase the amount of very long chain PUFAs. However, in a hybrid production system, the combination of male and female parents both containing very long chain PUFAs can yield a mid-parent amount of EPA+DPA+DHA, and in some cases even lower EPA+DPA+DHA than either parent.

Surprisingly, when hybrids were produced using a female heterozygous for the three QTL as previously described (and a male that is homozygous, heterozygous, or lacking all three QTL described herein, but all plants contain one or more tDNAs as described herein), the F1 hybrid seed demonstrated heterosis, that is it yielded higher EPA+DPA+DHA than either of the parents. In this case, the female alone produced 18.7% EPA+DPA+DHA, the male produced 13.6-14.8% EPA+DPA+DHA (in this case the male did not have the three QTL as described herein), and the F1 hybrid made from this cross produced 20.2-24.8% EPA+DPA+DHA. The same effect was observed when the female was heterozygous for the three QTL and the male was homozygous for the three QTL. In this case, the female alone produced 18.7% EPA+DPA+DHA, the male produced 13.8-14.5% EPA+DPA+DHA, and the F1 hybrid made from this cross produced 18.8-21.8% EPA+DPA+DHA.

BIBLIOGRAPHY

Clarke et al. A high-density SNP genotyping array for Brassica napus and its ancestral diploid species based on optimised selection of single-locus markers in the allotetraploid genome. Theor Appl Genet (2016) 129:1887-1899.

Endelman, J. B. Ridge regression and other kernels for genomic selection with R package rrBLUP. Plant Gen. (2011) 4(3):250-255.

Lipka, A., F. Tian, Q. Wang, J. Peiffer, M. Li, P. Bradbury, M. Gore, E. Buckler, and Z. Zhang. GAPIT: genome association and prediction integrated tool. Bioinformatics (2012) 28: 2397-2399

R Development Core Team. R: a language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria. (2015) http://www.R-project.org (accessed 31 Jul. 2016).

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the definition of a term incorporated by reference conflicts with a term defined herein, this specification shall control.