WO2016089677A2 - Novel qtl of brassica plant correlated to fatty acid profile - Google Patents

Novel qtl of brassica plant correlated to fatty acid profile Download PDF

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Publication number
WO2016089677A2
WO2016089677A2 PCT/US2015/062420 US2015062420W WO2016089677A2 WO 2016089677 A2 WO2016089677 A2 WO 2016089677A2 US 2015062420 W US2015062420 W US 2015062420W WO 2016089677 A2 WO2016089677 A2 WO 2016089677A2
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qtl8
qtl9
qtl3a
qtl4b
qtl5b
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PCT/US2015/062420
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French (fr)
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Richard Fletcher
David Herrmann
Honggang Zheng
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Cargill, Incorporated
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Publication of WO2016089677A2 publication Critical patent/WO2016089677A2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola
    • A01H6/202—Brassica napus [canola]

Definitions

  • This disclosure relates to Brassica plants and, more particularly, Brassica plants having one or more mutations at a novel quantitative trait locus denoted "QTL 1 " that correlates to the fatty acid profile of their seed oil.
  • the Brassica plants may also contain a modified fatty acyl-acyl carrier protein thioesterase A2 (fatAI) locus (e.g. , QTL3A), one or more modified fatty acid desaturase (fad2) loci (e.g. , QTL4A,and QTL4B), one or more modified fatty acyl-acyl carrier protein thioesterase B (fatB) loci (e.g.
  • the Brassica plants may further comprise three, or four modified fad3 loci (e.g. , QTL6A, QTL6B, QTL6C, and QTL6D).
  • the sequences for this application are shown in Table
  • saturated fat intake should be no more than 10 percent of total calories. Based on a 2,000-calorie-a-day diet, this is about 20 grams of saturated fat a day. While canola oil typically contains only about 7% to about 8% saturated fatty acids, a decrease in its saturated fatty acid content would improve the nutritional profile of the oil.
  • QTLs are regions of the genome which explain variation in continuously distributed phenotypes, and almost always contain many (hundreds or more) genes (Mackay, 2001 ).
  • Construction of a genetic map can be accomplished by analyzing the segregation of alleles in a recombinant population (Haldane, 1919).
  • a recombinant population of plants is often generated from a directed cross between two inbred lines, hereafter known as the parents. Recombination during meiosis results in a population of individuals whose genotypes are a mosaic of the parental alleles.
  • Parental alleles can be tracked via a number of marker methods including morphology (Sturtevant, 1913; Sax, 1923), isozyme (Hunter and Markert, 1957) and DNA (Botstein et al., 1980; Nakamura et al., 1987).
  • a DNA marker is simply a DNA sequence that shows sequence polymorphism among individuals within a species (Andersen and Lubberstedt, 2003). Analysis of the cosegregation of marker polymorphisms (alleles) within the recombinant population allows a researcher to construct a map of the estimated locations of each allele relative to others. Marker alleles that are found together more often are assumed to be located closer together because of a limitation in crossover events due to physical linkage (Muller, 1 916). Pairwise comparisons of the frequency of each allelic combination are used to create a genetic map which provides a visual illustration of their location on an organism's chromosomes.
  • QTL mapping is essentially the association of a locus with variation in a quantitative phenotype (Mauricio, 2001 ). Two pieces of information are needed for a QTL mapping experiment. First, genotype data for each individual within the recombinant population at each marker locus is recorded as the parental allele from which it originates. Second, phenotype data for the target trait is measured for each individual of the recombinant population. Statistical association analyses of the phenotype data and the marker data will reveal loci which explain a significant proportion of the variation in the phenotype data. The association between genetic map positions and a phenotype can then be inferred. The final product is a map of those areas of the genome containing QTLs.
  • plants including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), comprising one or more mutations in the QTL 1 locus, a novel quantitative trait locus identified in a doubled haploid (DH) population of 225 lines derived from a cross between I MC 106RR (Cargill, Incorporated, National Registration No. 5 1 1 8) and the biennial variety Wichita (Rife et al., 2001 ; Registration No. CV- 1 9, PI 612846). Also provided herein are methods of generating such plants and seeds, oils derived from such plants and seeds, and methods of making these oils.
  • DH doubled haploid
  • the QTL 1 locus is defined by its correlation to variations in the fatty acid profi le (e.g., C 1 8:0, C20:0, C22:0, and/or total saturates) of seed oil from the DH cross lines, and by the SNP markers identified herein.
  • the QTL 1 locus is believed to reside upon Brassica napus chromosome 15. However, it is understood that the locus is defined not by chromosomal position but by its SNP markers and its contribution to the fatty acid content of seed oil, and therefore the locus may appear on other chromosomes, particularly in progeny.
  • seeds of plants comprising one or more mutations in QTL 1 may yield oil having a total saturated fatty acid content of about 8.5% or less (e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5%).
  • the palmitic acid content of the oil may be about 3.0% to about 5.5% (e.g. , about 3.0% to about 5.5%, about 3.0% to about 5%, or about 3.5% to about 4.5%).
  • the stearic acid content of the oil may be about 1 .0% to about 3.5% (e.g. , about 1.0% to about 3.0%, about 1.0% to about 2.0%, or about 1.3% to about 2.0%)
  • the arachidic acid i.e.
  • eicosanoic acid content of the oil may be about 0.5% to about 1.5% (e.g. , about 0.5% to about 1 .2%, about 0.5% to about 1 .0%, or about 0.6% to about 1.0%).
  • the docosanoic acid content of the oil may be about 0.3% to about 0.8% (e.g. , about 0.3% to about 0.7%, about 0.4% to about 0.7%, or about 0.4% to about 0.65%).
  • the oil may have an oleic acid content of about 62% to about 85% or higher (e.g.
  • a linoleic acid content of about 8% to about 10%, and/or an a-linolenic acid content of no more than about 4% (e.g., about 2% to about 4%).
  • the plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein comprising one or more mutations in QTL 1 (N 1 5) may further comprise ( 1 ) one or more QTLs and/or known genetic variants (mutations) selected from the group consisting of: falA2 mutations (e.g. , QTL3A),fad2 mutations (e.g. , QTL4A and QTL4B),/a/5 mutations (e.g., QTL5A, QTL5B, QTL5C, and QTL5D), kasIII mutations (e.g. , QTL7), mutations on the chromosome N l (e.g.
  • plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein comprising one or more mutations in QTL1 may further comprise one or more mutant alleles at:
  • plants including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein may further comprise one or more mutant al leles at:
  • plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein comprising one or more mutations in QTL1 locus may further comprise one or more mutant alleles at:
  • plants including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein may further comprise one or more mutant alleles at:
  • plants including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein comprising one or more mutations in QTL1 and one or more of fatA2 mutations, one or more fatB mutations, and/or one or more mutations in QTL7 and/or QTL8 and/or QTL9 as described above may produce oils with a total saturated fatty acid content less than about 8.5% (e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5% total saturates), oils having less than about 3.6% saturated fatty acids (e.g.
  • plants including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein comprising one or more mutations in
  • QTL 1 and one or more mutations at one or more loci selected from the group consisting of fatA2 locus (e.g. , QTL3A),fatB loci (e.g., QTL5A, QTL5B, QTL5C, and QTL5D), kasIII locus (e.g. , QTL7), loci on the chromosome 1 (e.g. , QTL8), and loci on the chromosome N 19 (e.g., QTL9) may further comprise mutations at one or more fad2 alleles (e.g. , QTL4A and QT43B) and/or three or more fad3 alleles (e.g.
  • the presence or absence of specific combinations of mutations at fad2 and/or fad3 alleles may be used to tailor the oleic acid and a-linolenic acid content of oils produced by the plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), to the desired end use of the oil.
  • the present disclosure provides Brassica plants (e.g. , Brassica napus, Brassica juncea, Brassica rapa, Brassica oleracea, or Brassica carinata), including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), that comprise one or more mutations in QTL 1 on chromosomes N l 5 relative to the wild-type allele in
  • Plants comprising mutations in QTL 1 can be derived from IMC 106RR either as the result of a cross in which IMC 106RR is directly involved, or by using plants derived from IMC 106RR comprising all or part of the genome corresponding to QTL 1 .
  • Plants bearing the region of IMC 106RR corresponding to QTL1 can be DH plant(s) derived from a cross between IMC106RR, Fi hybrids of IMC 106RR, or F2, F3, F4 or subsequent generations of progeny derived from IMC 106RR.
  • Such plants may further comprise one or more mutations at one, two, three, four, five, six, seven, eight, nine, or ten selected from the group consisting of QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL7, QTL8, and QTL9.
  • Brassica plants including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), comprising one or more mutations in QTL 1 further comprise ( 1 ) one or more mutant alleles at three, or four, of different loci selected from the group consisting of QTL6A, QTL6B, QTL6C, and QTL6D; and/or (2) one or more modified (e.g.
  • QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D,QTL 7, QTL8, and QTL9 are the alleles recited in Table C.
  • methods are provided herein for producing oil from the Brassica plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), disclosed herein.
  • these methods comprise ( 1 ) crushing seeds produced from at least one Brassica plant comprising one or more mutations in QTL1 that contributes to the reduced saturated fatty acid content in IMC 106RR described herein, and one or more mutations selected from the group consisting of fatA2 mutations (e.g. , QTL3A), fad2 mutations (e.g.
  • the oil further may further comprise about 1 .6% to about 2.3% eicosenoic acid, about 78% to about 80% oleic acid, about 8% to about 10% linoleic acid, and/or about 2% to about 4% a-linolenic acid.
  • Brassica plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), used in these methods may further comprise three, or four, fad3 mutations (e.g. , QTL6A, QTL6B, QTL6C, and QTL6D).
  • these methods comprise:
  • said first Brassica parent plants comprise a nucleic acid sequence having greater than 80% identity (e.g. , 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.8%, 99.9% or 99.99%) to all or part of the genomic sequences between the chromosome N l 5 (QTL 1 ) SNP markers at positions 43730765 and 45730765 of the B. napus line IMC 1066RR, wherein said part of the genomic sequences of the B.
  • napus IMC 106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides, wherein said first parent plant optionally comprises one or more mutant alleles at one, two, three, four or more different loci selected from the group consisting offatA2 locus (e.g. , QTL3A), fad2 loci (e.g. , QTL4A and QTL4B), fatB loci (e.g., QTL5A, QTL5B, QTL5C, and QTL5D), kasIII locus (e.g. , QTL7), locus on the chromosome N 1 (e.g. , QTL8), and locus on the chromosome N 19 (e.g.
  • fatA2 locus e.g. , QTL3A
  • fad2 loci e.g. , QTL4A and QTL4B
  • fatB loci e.g., QTL5A, QTL5B
  • said one or more second Brassica parent plants optionally comprise a mutant allele at one, two, three, four or more different loci selected from the group consisting of fatA2 locus (e.g., QTL3A),fa 2 loci (e.g. , QTL4A and QTL4B),fatB loci (e.g., QTL5A, QTL5B, QTL5C, and QTL5D), kasIII locus (e.g., QTL7), locus on the chromosome N l (e.g. , QTL8), and locus on the chromosome N l 9 (e.g. , QTL9) of said first Brassica parent;
  • fatA2 locus e.g., QTL3A
  • fa 2 loci e.g. , QTL4A and QTL4B
  • fatB loci e.g., QTL5A, QTL5B, QTL5C, and QTL5D
  • kasIII locus
  • said mutant alleles at one, two, three, four or more different loci selected from the group consisting of fatA2 locus (e.g. , QTL3 A), fad2 loci (e.g. , QTL4A and QTL4B), fatB loci (e.g., QTL5A, QTL5B, QTL5C, and QTL5D), kasIII locus (e.g., QTL7), locus on the chromosome N l (e.g. , QTL8), and locus on the chromosome N l 9 (e.g. , QTL9), present in said first and/or second Brassica parent if present in said first or second parent,
  • fatA2 locus e.g. , QTL3 A
  • fad2 loci e.g. , QTL4A and QTL4B
  • fatB loci e.g., QTL5A, QTL5B, QTL5C, and QTL5
  • the present disclosure further includes and provides for methods of selecting Brassica plants for the presence or absence of all or part of QTL 1 of IMC 106RR (Cargill,
  • Such methods of selecting or breeding Brassica plants comprise obtaining one or more Brassica plants and assessing their DNA to determine the presence or absence of QTL1 (on chromosome N 1 5). Based upon the results of the assessment, plants are selected for the presence or absence of all or part of QTL 1 to produce one or more selected plants.
  • this disclosure provides ( 1 ) canola oil having an oleic acid content of about 78% to about 80%, a linoleic acid content of about 8% to about 10%, an a- linolenic acid content of no more than about 4%, and an eicosenoic acid content of about 1.6% to about 2.3%; (2) canola oil having a total saturated fatty acid content less than about 8.5% (e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5% total saturates), and (3) canola oil having less than about 3.6% saturated fatty acids ⁇ e.g.
  • this disclosure provides canola oil having a total saturated fatty acid content of no more than about 3.7% and an oleic acid content of about 62% to about 85% (e.g. , about 62% to about 65%, about 65% to about 72%, about 72% to about 75%, about 75% to about 80%, about 80% to about 84% and/or about 82% to about 85%).
  • the oil may further comprise about 3.6% to about 5.2% of palmitic acid, and/or about 1.3% to about 3.0% of stearic acid.
  • the oil may further comprise about 0.60% to about 1.10% of arachidic acid, about 0.40% to about 0.65% of docosanoic acid, and/or about 1 .6% to about 1 .9% of eicosenoic acid.
  • the total saturated fatty acid content may be about 3.4% to about 3.7%.
  • the Brassica plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), disclosed herein are not genetically modified (i.e., they are non-transgenic), making them particularly useful for producing canola oils for use in food applications. In other embodiments, they may comprise only transgenes for herbicide tolerance.
  • the term "plant” or “plants” includes parts thereof and progeny, i.e. , descendants of a particular plant or plant line, as well as cells or tissues from the plant, unless stated otherwise.
  • Parts of plants include, but are not limited to, any one or more of: a leaf, pollen, an ovule, an embryo, a cotyledon, a hypocotyl, a meristematic cell, callus, a microspore, a root, a root tip, a pistil, an anther, a flower, a seed, a shoot, a stem, a pod, petiole and a cell or protoplast of any thereof.
  • Progeny of an instant plant include seeds formed on Fi , F 2 , F3, F4 and subsequent generation plants, or seeds formed on BC
  • allele or “alleles” refers to one or more alternative forms of a locus.
  • mutant alleles or “mutation” of alleles include alleles having one or more mutations, such as insertions, deletions, stop codons, base changes (e.g. , transitions or transversions), or alterations in splice junctions, which give rise to altered gene products. Modifications in alleles may arise in coding or non-coding regions (e.g. , promoter regions, exons, introns or splice junctions).
  • a "line” is a group of plants that display little or no genetic variation between individuals for at least one trait. Such lines may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques.
  • variable refers to a line which is used for commercial production, and includes hybrid varieties and open-pollinated varieties.
  • 24:0 or C24:0 refers to lignoceric acid
  • 22:0 or C22:0 refers to behenic acid
  • 20:0 or C20:0 refers to arachidic acid
  • 1 8:0 or C 1 8:0 refers to stearic acid
  • 16:0 or C 16:0 refers to palmitic acid
  • 14:0 or C 14:0 refers to myristic acid, the terminal carboxyl groups of any of which may or may not be esterified unless indicated otherwise.
  • total saturated fatty acid content refers to the total of myristic acid (C 14:0), palmitic acid (C 16:0), stearic acid (C 1 8:0), arachidic acid (C20:0), behenic acid (C22:0), and lignoceric acid (C24:0).
  • total polyunsaturates refers to the total of linoleic acid and a-linolenic acid.
  • Transgenic or “genetically modified organisms” as used herein are organisms whose genetic material has been altered using techniques generally known as "recombinant DNA technology".
  • Recombinant DNA technology is the ability to combine D A molecules from different sources into one molecule ex vivo (e.g. , in a test tube). This terminology generally does not cover organisms whose genetic composition has been altered by conventional cross-breeding or by "mutagenesis” breeding, as these methods predate the discovery of recombinant DNA techniques. See, World Health Organization, Biorisk management Laboratory biosecurity guidance, 2006 World Health Organization
  • Non-transgenic refers to plants and food products derived from plants that are not “transgenic” or “genetically modified organisms” as defined above.
  • the plants described herein are non-transgenic to the extent that they are derived by mutagenesis.
  • position refers to the location of the SNP by base pair within the pseudo molecules of Brassica oleracea genome (B. oleracea TO 1000 version 4; released date is 12-Jan-2012) obtained from Canseq consortium (http://aafc- aac.usask.ca/canseq/).
  • weight percent refers to the percent by weight of the fatty acids having from 14 carbon atoms (C 14 fatty acids) to 24 carbon atoms (C24 fatty acids).
  • percent by weight refers to the percent by weight of the total of those fatty acids in the seed oil fraction.
  • sequence identity refers to the degree of identity between any given nucleic acid sequence and a target nucleic acid sequence. Percent sequence identity is calculated by determining the number of matched positions in aligned nucleic acid sequences, dividing the number of matched positions by the total number of aligned nucleotides, and multiplying by 100. A matched position refers to a position in which identical nucleotides occur at the same position in aligned nucleic acid sequences. Percent sequence identity also can be determined for any amino acid sequence.
  • a target nucleic acid or amino acid sequence is compared to the identified nucleic acid or amino acid sequence using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN and BLASTP.
  • This stand-alone version of BLASTZ can be obtained from Fish & Richardson's web site (World Wide Web at fr.com/blast) or the U.S. government's National Center for Biotechnology Information web site (World Wide Web at ncbi.nlm.nih.gov). Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ.
  • BI2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g. , C: ⁇ seq l .txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g. , C: ⁇ seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g.
  • C ⁇ output.txt
  • -q is set to - 1
  • -r is set to 2; and all other options are left at their default setting.
  • the following command will generate an output file containing a comparison between two sequences: C: ⁇ B12seq -i c: ⁇ seql .txt -j c: ⁇ seq2.txt -p blastn -o c: ⁇ output.txt -q - 1 -r 2. If the target sequence shares homology with any portion of the identified sequence, then the designated output file will present those regions of homology as aligned sequences. If the target sequence does not share homology with any portion of the identified sequence, then the designated output file will not present aligned sequences.
  • a length is determined by counting the number of consecutive nucleotides from the target sequence presented in alignment with the sequence from the identified sequence starting with any matched position and ending with any other matched position.
  • a matched position is any position where an identical nucleotide is presented in both the target and identified sequences. Gaps presented in the target sequence are not counted since gaps are not nucleotides. Likewise, gaps presented in the identified sequence are not counted since target sequence nucleotides are counted, not nucleotides from the identified sequence.
  • the percent identity over a particular length is determined by counting the number of matched positions over that length and dividing that number by the length followed by multiplying the resulting value by 100.
  • mapping of QTL1 has been accomplished using the DH population of 225 lines derived from a cross between IMC 106RR and the biennial variety Wichita. Mapping in the population permits localization of QTL 1 on chromosome N 15 that explains a significant proportion of the variation in C 18:0, C20:0, C22:0 and total saturates (Table 1 ). DH lines carrying the IMC 106RR allele are significantly lower in each fatty acid component than DH lines carrying the Wichita al lele. Analysis of the QTL 1 region using the single marker regression approach identifies the marker located at 42605157 bp on C05 of the C genome as the most highly correlated (the highest percent variance explained (R 2 ), Table 2, based on a C genome assembly).
  • the QTL 1 interval is defined as spanning from 43730765 ⁇ 15730765 bp on N 15 of the DH 12075 B. napus genome assembly.
  • Geno sequencing data e.g., fastq files
  • a Brassica napus reference genome (19 linkage groups of B. napus genotype DH 12075, CanSeq Consortium)
  • SeqMan NGen v4 DNAStar, Madison, WI
  • the alignment is performed using default settings for read mapping and SNP calling.
  • a list of 38 SNPs is generated (Table 4), requiring all selected SNPs to be homozygous variant and to be unique to the IMC 106R.R genotype (i .e., different than the Wichita sequence and the DH 12075 reference sequence).
  • the Brassica plants provided herein are non-transgenic. In other embodiments, they may comprise only transgenes for herbicide tolerance.
  • Examples of the Brassica plants include, without limitation, B. napus, B. juncea, B. rapa, B. oleracea, and B. carinata.
  • the Brassica plants yield seed oil with a total saturated fatty acid content of about 7% or less, or having a total saturated fatty acid content of about 3.6% or less, that comprise one or more mutations at a locus termed QTL I on chromosome N l 5. This locus is defined by its correlation to the variation of C 18:0 and/or C20:0 and/or C22:0 fatty acid content of seed oil and the SNP markers identified herein.
  • QTL I is believed to reside upon B. napus chromosome N l 5 based upon the mapping populations described herein. However, it is understood that the locus is defined not by chromosomal position but by its SNP markers and its correlation to the fatty acids content of seed oil, and therefore the locus may appear on other chromosomes, particularly in progeny. The appearance of QTL I on other
  • chromosomes may result from a variety of events including, but not limited to, homologous chromosomal crossover events.
  • the occurrence of crossover events may be higher in plants such as B. napus, which is an allopolyploid species.
  • a Brassica plant or a part thereof comprising a nucleic acid sequence having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences between the chromosome N 1 5 (QTL 1 ) SNP markers at positions 43730765 and 45730765 of the B. napus line 1 C 106RR, wherein said part of the genomic sequences of the B.
  • napus 1MC 106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
  • the Brassica plant or a part thereof is transgenic; in certain embodiments, the Brassica plant or a part thereof is non-transgenic; and in other embodiments, the Brassica plant or a part thereof comprises only transgenes for herbicide tolerance.
  • the Brassica plant, or a part thereof comprises a nucleic acid sequence having greater than about 80% (e.g., about 85%), about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%), about 99.995%, or about 99.999%) identity to all or part of the genomic sequence of the B.
  • napus line IMC 106RR between positions 43730765 and 45730765 and/or any two SNP markers between those positions, wherein the SNP markers between those positions are selected from: 43849348, 43877053, 44223 135, 44252880, 44407747, 44617229, 44659768, 44736857, 44746871 , 44756604, 44771673, 44794771 , 44796526, 44798855, 44839954, 44855978, 44859545, 44872594, 44923355, 44985743, 45007425, 45093354, 45147723, 45159244, 45165499, 45256038, 45260347, 45278355, 45298286, 45312516, 45354975, 45365977, 45378346, 45402371 , 45409080, 45496931 , 45499138, and 4572071 5, wherein said part of the genomic sequences of the B. napus I C 106RR
  • the Brassica plants comprise one, two, three, four, five, ten, fifteen or more QTL 1 markers selected from the group consisting of:
  • the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%)) identity to all or part of the genomic sequences within the segments between any two chromosome N l 5 (QTL 1 ) SNP markers at positions selected from: 43849348, 43877053, 44223135, 44252880, 44407747, 44617229, 44659768, 44736857, 44746871 , 44756604, 44771673, 44794771 , 44796526, 44798855, 44839954, 44855978, 44859545, 44872594, 44923355, 44985743, 45007425, 45093354, 45147723, 45159244, 45165499, 45256038, 45
  • napus line IMC 106RR (Cargill, Incorporated, National Registration No. 51 1 8), wherein said part of the genomic sequences of the B. napus IMC 106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
  • the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences comprising any one or more (e.g., two, three, or four) chromosome N l 5 (QTL 1 ) SNP markers at positions selected from: 43849348, 43877053, 44223135, 44252880, 44407747, 44617229, 44659768, 44736857, 44746871 , 44756604, 44771673, 44794771 , 44796526, 44798855, 44839954, 44855978, 44859545, 44872594, 44923355, 44985743, 45007425, 45093354, 45147723, 451 5
  • napus line IMC 106RR (Cargill, Incorporated, National Registration No. 51 18), wherein said part of the genomic sequences of the B. napus IMC 106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
  • the nucleic acid comprises one or more sequences selected from the group consisting of SEQ ID NOs: 2- 1 , 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2- 10, 2- 1 1 , 2- 12, 2- 13, 2- 14, 2- 15, 2- 16, 2- 1 7, 2- 1 8, 2- 19, 2-20, 2-21 , 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31 , 2-32, 2-33, 2-34, 2- 35, 2-36, 2-37, and 2-38.
  • the Brassica plants comprise a nucleic acid sequence having greater than 80% identity to all or part of the genomic sequences between the chromosome N l 5 (QTL 1 ) SNP markers at positions 43849348 and 4572071 5 of the genomic sequence of B. napus IMC 106RR line, wherein said part of the genomic sequences of the B. napus IMC 106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
  • the Brassica plants comprise one, two, or three single nucleotide polymorphisms on B. napus chromosome N 1 5 selected from the group consisting of C to T transitions identified in the B. napus I MC 106RR line, National Registration No. 51 18, at locations: 43877053, 44659768, and 44746871 .
  • the Brassica plants comprise one, two, or three single nucleotide polymorphisms on B. napus chromosome N l 5 selected from the group consisting of T to C transitions identified in the B. napus IMC 106RR line, National Registration No. 51 18, at locations: 45093354, 45354975, and 45378346.
  • the Brassica plants comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve single nucleotide polymorphisms on B. napus chromosome N l 5 selected from the group consisting of G to A transitions identified in the B. napus IMC 106RR line, National Registration No. 51 1 8, at locations: 44252880, 44407747, 44617229, 44756604, 44794771 , 44855978, 44859545, 44872594, 45007425, 45 159244, 453125 16, and 45365977.
  • the Brassica plants comprise one, two, three, four, five, or six single nucleotide polymorphisms on B. napus chromosome N l 5 selected from the group consisting of A to G transitions identified in the B. napus IMC 106RR line, National Registration No. 51 1 8, at locations: 44617229, 44796526, 44923355, 45147723, 45260347, and 45278355.
  • the Brassica plant comprises a B. napus N 15 chromosome having greater than 95%, 97.5%, 98%, 99%, 99.9%, 99.99%, 99.999% identity or having 100% identity to all or part of the genomic sequences of the B. napus I MC 106RR line, National Registration No. 51 18, on an N 15 chromosomal segment selected from the group consisting of segments:
  • SNP 43849348 and ending with SNP 4572071 5 or SNP 45402371 beginning with SNP 43849348 and ending with SNP 45312516 or SNP 45256038; beginning with SNP 43849348 and ending with SNP 45165499 or SNP 45007425; beginning with SNP 43849348 and ending with SNP 44923355 or SNP 44839954; beginning with SNP 43849348 and ending with SNP 44771673 or SNP 44659768; beginning with SNP 43849348 and ending with SNP 44407747 or SNP 44223 1 35; beginning with SNP 44223 1 35 and ending with SNP 4572071 5 or SNP 45402371 ; beginning with SNP 44223 135 and ending with SNP 45312516 or SNP 45256038; beginning with SNP 44223135 and ending with SNP 45165499 or SNP 45007425; beginning with SNP 44223135 and ending with SNP 44923355 or SNP 44839954;
  • a number of candidate genes that may correlate to the variance of fatty acid profile (e.g., C 18:0 and/or C20:0 and/or C22:0 and/or total saturates) of plants are present in or tightly linked to the region into which QTL 1 has been mapped.
  • phosphatidylinositol-4-phosphate 5-kinase family protein (AT3G 14270); ( 1 1 ) FAD- dependent oxidoreductase family protein (AT3G 10370); ( 12) phosphatidyl inositol monophosphate 5 kinase (AT3G09920); ( 1 3) phosphatidic acid phosphohydrolase 1 (AT3G09560); ( 14) lipid transfer protein 6 (AT3G08770); ( 1 5) phospholipase C 2
  • A3G08510 6-phosphogluconate dehydrogenase family protein (AT3G07690); ( 17) bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin superfamily protein (AT3G07450) and ( 18) lipase class 3 family protein (AT3G07400) are among the genes present in the interval, which, as previously indicated, is believed to be on chromosome N 1 5, onto which QTL 1 has been mapped.
  • Brassica plants described herein may produce a seed oil having a total saturated fatty acid content of about 2.5% to about 5.5%, about 3% to about 5%, about 3% to about 4.5%, about 3.25% to about 3.75%, about 3% to about 3.5%, about 3.6% to about 5%, about 4% to about 5.5%, or about 4% to about 5%.
  • Oi ls having total saturated fatty acid content of about 8.5% or lower are perceived to have improved nutritional quality and can help consumers reduce their intake of saturated fatty acids.
  • Brassica plants having one or more mutations at QTL 1 locus described herein can yield a seed oil having a total saturated fatty acid content of about 8.5% or lower (e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5% total saturates), in combination with about 60% to about 70%, about 71 % to about 80%, or more than about 80% oleic acid content.
  • Such Brassica plants can produce a seed oil having a fatty acid content tailored to the desired end use of the oil (e.g. , frying or other food applications).
  • Brassica plants can be produced that yield a seed oil having a total saturated fatty acid content of about 8.5% or lower, an oleic acid content of about 60% to about 70%, and an a-linolenic acid content of about 2% to about 5%.
  • Total polyunsaturates in such seed oi ls typically are less than about 30%, preferably less than about 28%, e.g. , about 1 5% to about 27.5%, about 17% to about 27%, or about 20% to about 25%.
  • Canola oils having such fatty acid contents are particularly useful for frying applications due to the polyunsaturated content, which is low enough to provide improved oxidative stability for frying, yet high enough to impart the desired fried flavor to the food being fried, and are an improvement over commodity type canola oils.
  • the fatty acid content of commodity type canola oils typically is about 6% to about 8% total saturated fatty acids, about 55% to about 65% oleic acid, about 22% to about 30% linoleic acid, and about 7% to about 10% a- linolenic acid.
  • Brassica plants described herein also may yield a seed oil having a total saturated fatty acid content of about 1.6% to about 3%, about 2% to about 4%, about 3% to about 6%, about 71% to about 80%, or about 2% to about 5.0%.
  • Canola oils having such fatty acid contents have higher oxidative stabilities than oils with a lower oleic acid and higher ⁇ -linolenic acid content and/or commodity type canola oils.
  • canola oils are useful for coating applications (e.g. , spray-coatings), formulating food products, or other applications where shelf-life stability is desired.
  • Brassica plants described herein can yield a seed oi l having a total saturated fatty acid content of less than about 8.5% ⁇ e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5% total saturates), an oleic acid content of about 81 % to about 90% and an ⁇ -linolenic acid content of about 2% to about 5%.
  • Canola oils having a total saturated fatty acid content less than about 8.5% ⁇ e.g.
  • an oleic acid content of about 81 % to about 90%, and an ⁇ -linolenic acid content of about 2% to about 5% are particularly useful for food applications requiring high oxidative stability and a reduced saturated fatty acid content.
  • the present disclosure further includes and provides for methods of selecting or breeding Brassica plants for the presence or absence of all or part of QTLI of IMC 106RR (Cargill, Incorporated, National Registration No. 51 1 8) that may be employed, for example, as molecular guided breeding programs.
  • Such methods of selecting or breeding Brassica plants comprise obtaining one or more Brassica plants and assessing their DNA to determine the presence or absence of all or part of QTLI (on chromosome NI5). Based upon the results of the assessment, plants are selected for the presence or absence of all or part of QTLI to produce one or more selected plants.
  • Such methods may be used, for example, to determine which progeny resulting from a cross have all or part of QTLI, and accordingly to guide preparation of plants having one or both of those QTLs in combination with other desirable genes/traits.
  • determining the presence of all or part of QTL I in plants comprises determining the presence of mutations appearing in IMC 106 R in the QTL 1 region that do not appear in its parent. Accordingly, plants can be selected by assessing them for the presence of one or more individual SNPs appearing in Table 4 for QTL I . Plants may also be assessed for larger portions of those QTL regions (e.g., regions encompassing one or more SNPs in Table 4).
  • plants may be selected by determining the presence of one, two, three, four, five, ten, fifteen or more QTL I markers selected from the group consisting of: 43849348, 43877053, 44223 135, 44252880, 44407747, 4461 7229, 44659768, 44736857, 44746871 , 44756604, 44771673, 44794771 , 44796526, 44798855, 44839954, 44855978, 44859545, 44872594, 44923355, 44985743, 45007425, 45093354, 45147723, 45159244, 45165499, 45256038, 45260347, 45278355, 45298286, 45312516, 45354975, 45365977, 45378346, 45402371 , 45409080, 45496931 , 45499138, and 45720715.
  • QTL I markers selected from the group consisting of: 43849348, 43877053, 44223
  • plants may be assessed to determine the presence or absence of QTLI chromosomal segments including a segment selected from the chromosomal regions:
  • plants may be assessed to determine the presence or absence of QTL 1 SNP.
  • Any suitable method known in the art may be used to assess plants to determine if they comprise all or part of QTL1 .
  • Some suitable methods include, but are not limited to, sequencing, hybridization assays, polymerase chain reaction (PCR), ligase chain reaction (LCR), and genotyping-by-sequencing (GBS).
  • the plants may be assessed for their fatty acid content. More specifically, plants may be assessed for their fatty acid profile (i.e., the types and/or relative amount of fatty acids they produce, typically in their seed) and their total fatty acid production. Among the fatty acids that can be examined are saturated fats (e.g., 16:0, 18:0, 20:0, etc.), monounsaturated fats, and polyunsaturated fats. Analysis of fatty acid profile and/or content may be directed to one or more selected plants (or their seed) selected and/or the progeny of such plants.
  • saturated fats e.g., 16:0, 18:0, 20:0, etc.
  • monounsaturated fats e.g., monounsaturated fats
  • polyunsaturated fats e.g., etc.
  • Analysis of fatty acid profile and/or content may be directed to one or more selected plants (or their seed) selected and/or the progeny of such plants.
  • the Brassica plants described herein comprise one or more alleles QTL 1 of the IMC 106RR line and further comprise a mutant allele for a fatty acyl-ACP thioesterase.
  • Fatty acyl-ACP thioesterases hydrolyze acyl-ACPs in the chloroplast to release the newly synthesized fatty acid from ACP, effectively removing it from further chain elongation in the plastid.
  • the free fatty acid can then leave the plastid, become bound to CoenzymeA (CoA) and enter the Kennedy pathway in the endoplasmic reticulum (ER) for triacylglycerol (TAG) biosynthesis.
  • CoA CoenzymeA
  • ER endoplasmic reticulum
  • TAG triacylglycerol
  • a method of DNA-assisted selection of a reduced-palmitic acid oi l trait in Brassica plants comprising:
  • the method of DNA-assisted selected described supra further comprises selecting a second one of the Brassica plants in which QTL 1 , or at least said part of QTL1 , is present.
  • the method of DNA-assisted selected described supra further comprises selecting a second one of the Brassica plants in which QTL 1 , or at least said part of QTL1 , is present, and:
  • the method of DNA-assisted selected described supra further comprises:
  • Brassica plants comprising reduced activities of certain enzymes related to the production of saturated and unsaturated fatty acids.
  • Fatty acyl-ACP thioesterases hydrolyze acyl-ACPs in the chloroplast to release the newly synthesized fatty acid from ACP, effectively removing it from further chain elongation in the plastid.
  • the free fatty acid can then leave the plastid, become bound to CoenzymeA (CoA) and enter the Kennedy pathway in the endoplasmic reticulum (ER) for triacylglycerol (TAG) biosynthesis.
  • CoA CoenzymeA
  • ER endoplasmic reticulum
  • TAG triacylglycerol
  • FATA2 prefer oleoyl (C 18: 1 ) ACP substrates with minor activity towards 18:0 and 16:0 ACPs, while members of the FATB family (e.g., FATB) hydrolyze primarily saturated acyl-ACPs between 8 and 1 8 carbons in length.
  • members of the FATB family e.g., FATB
  • FATB hydrolyze primarily saturated acyl-ACPs between 8 and 1 8 carbons in length.
  • Reduced activities, including absence of detectable activity, of FATA2, and/or FATB may be achieved by modifying an endogenous fatA2 and/or fatB allele(s), respectively.
  • An endogenous fatA2 or fat3B allele can be modified by, for example, mutagenesis or by using homologous recombination to replace an endogenous plant gene with a variant containing one or more mutations (e.g. , produced using site-directed mutagenesis). See, e.g. , Townsend et al., Nature 459:442-445 (2009); Tovkach et al., Plant J, 57:747-757 (2009); and Lloyd et al., Proc. Natl. Acad. Sci.
  • mutant alleles can be modified by mutagenesis or by using homologous recombination to replace an endogenous gene with a variant. Alleles comprising mutations obtained through mutagenesis are referred to as mutant alleles herein.
  • Reduced activities can be inferred from the decreased level of saturated fatty acids in the seed oil compared with the seed oil from a control plant.
  • the Brassica l ine Topas ATCC deposit 40624, may serve as a control plant.
  • B. napus cv. Topas As a reference, it has been redeposited and designated ATCC deposit PTA- 120738.
  • reduced activities can be assessed in plant extracts using assays for fatty acyl-ACP hydrolysis. See, for example, Bon Rush et al., Plant Cell 15: 1020- 1033 (2003); and Eccleston and Ohlrogge, Plant Cell 10:613-622 (1998).
  • mutagenic agents include, for example, ethyl methane sulfonate (EMS), methyl N-nitrosoguanidine (MN G), ethidium bromide, diepoxybutane, ionizing radiation, x-rays, UV rays and other mutagens known in the art.
  • EMS ethyl methane sulfonate
  • MN G methyl N-nitrosoguanidine
  • ionizing radiation x-rays
  • UV rays UV rays
  • a combination of mutagens such as EMS and MNNG, can be used to induce mutagenesis.
  • the treated population, or a subsequent generation of that population can be screened for specific enzyme activities (e.g., reduced thioesterase or acyltransferase activity) that result from the mutation(s), e.g. , by determining the fatty acid profile of the population and comparing it to a corresponding non-mutagenized population.
  • Mutations can be in any portion of a gene, including coding sequence, intron sequence and regulatory elements, that renders the resulting gene product non-functional or with reduced activities. Such mutations can lead to deletion or insertion of amino acids, and conservative or non- conservative amino acid substitutions in the corresponding gene product.
  • the mutation is a nonsense mutation, which results in the introduction of a stop codon (TGA, TAA, or TAG) and production of a truncated polypeptide.
  • the mutation is a splice site mutation which alters or abolishes the correct splicing of the pre-mRNA sequence, resulting in a protein of different amino acid sequence than the wild type. For example, one or more exons may be skipped during RNA splicing, resulting in a protein lacking the amino acids encoded by the skipped exons.
  • the reading frame may be altered by incorrect splicing, one or more introns may be retained, alternate splice donors or acceptors may be generated, splicing may be initiated at an alternate position, or alternative polyadenylation signals may be generated.
  • more than one mutation or more than one type of mutation is introduced.
  • Insertions, deletions, or substitutions of amino acids in a coding sequence may, for example, disrupt the conformation of essential alpha-helical or beta-pleated sheet regions of the resulting gene product.
  • Amino acid insertions, deletions, or substitutions also can disrupt binding, alter substrate specificity, or disrupt catalytic sites important for gene product activity. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product non-functional, compared to a smaller number of inserted or deleted amino acids.
  • Non-conservative amino acid substitutions may replace an amino acid of one class with an amino acid of a different class.
  • Non-conservative substitutions may make a substantial change in the charge or hydrophobicity of the gene product.
  • Non-conservative amino acid substitutions may also make a substantial change in the bulk of the residue side chain, e.g. , substituting an alanine residue for an isoleucine residue.
  • non-conservative substitutions include the substitution of a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid. Because there are only 20 amino acids encoded in a gene, substitutions that result in reduced activity may be determined by routine experimentation, incorporating amino acids of a different class in the region of the gene product targeted for mutation.
  • mutations may arise in the non- coding portions of genes, such as the promoter regions that alter the plants ability to express normal amounts of the mRNA and the protein it encodes. Mutations may also arise that increase the activity of proteins such as, for example, mutations that favor the pathways consistent with the saturated fatty acid profiles described herein for Brassica plants bearing QTL1.
  • the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus I MC 106RR and further comprise one or more mutant alleles at one fatA2 locus, wherein the one or more mutant alleles result in the production of a FATA2 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2 polypeptide (SEQ ID NO: QTL3A.p, wherein the position corresponding to position 255 as numbered based on the alignment to the Arabidopsis thaliana FATA2 polypeptide set forth in GenBank Accession No. NP_1 93041 .1 , protein (SEQ ID NO:2.p); GenBank Accession No. NM_1 1 7374, mRNA is leucine).
  • the mutant allele may comprise a nucleic acid that encodes a FATA2 polypeptide having a non-conservative substitution within a helix/4-stranded sheet (4HBT) domain (also referred to as a hot-dog domain) or a non-conservative substitution of a residue affecting catalytic activity or substrate specificity.
  • 4HBT helix/4-stranded sheet
  • the mutant allele at the fatA2 locus comprises a nucleic acid encoding a FATA2 polypeptide having a substitution in a region (SEQ ID: QTL3A.p, wherein the position corresponding to position 255 as numbered based on the alignment to the Arabidopsis thaliana FATA2 polypeptide set forth in GenBank Accession No. NP_193041 .1 , protein (SEQ ID NO:2.p); GenBank Accession No.
  • NM_1 17374, mRNA is leucine) of the polypeptide corresponding to residues 242 to 277 of the FATA2 polypeptide (as numbered based on the alignment to the Arabidopsis thaliana FATA2 polypeptide set forth in GenBank Accession No. NP_193041 . 1 , protein (SEQ ID NO:2.p); GenBank Accession No. NM_1 17374, mRNA).
  • Examples of such a nucleic acid include, without limitation, the nucleic acid sequences of SEQ ID NO: QTL3A.n, wherein nucleotide 1387 is T. This region of FATA2 is highly conserved in Arabidopsis and Brassica.
  • SEQ ID: QTL3A.p sets forth the predicted amino acid sequence of the Brassica FATA2 polypeptide.
  • the FATA2 polypeptide can have a substitution of a leucine residue for proline at the position corresponding to position 255 of the Arabidopsis FATA2 polypeptide (i.e. , SEQ ID NO: 2.p and QTL3A.p).
  • the proline in the B. napus sequence corresponding to position 255 in Arabidopsis is conserved among B. napus, B. rapa, B.
  • juncea Zea mays, Sorghum bicolor, Oryza sativa Indica (rice), Triticum aestivum, Glycine max, Jatropha (tree species), Carthamus tinctorius, Cuphea hookeriana, Iris tectorum, Perilla frutescens, Helianthus annuus, Garcinia mangostana, Picea sitchensis, Physcomitrella patens subsp. Patens, Elaeis guineensis, Vitis vinifera, Elaeis oleifera, Camellia oleifera, Arachis hypogaea, Capsicum annuum, Populus trichocarpa, and
  • Diploknema butyracea The mutation at position 255 is associated with a phenotype producing an oil with a total saturated fatty acid content of about 8.5% or lower (e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5% total saturates), stearic acid content of about 4.0% or lower, arachidic acid content of about 1 .5% or lower, and an increased eicosenoic acid phenotype.
  • the stearic acid content phenotype is negatively correlated with the eicosenoic acid phenotype.
  • the loci have at least about 90% (e.g. , at least about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9% or about 99.99%) sequence identity to the nucleotide sequence set forth in QTL3A
  • the nucleotide sequences set forth in QTL3A are representative nucleotide sequences from the fatA2 gene from B. napus line 15.24.
  • Fatty acyl-ACP thioesterase B (FATB).
  • Brassica napus contains 6 different fatB isoforms (i.e. , different forms of the FATB polypeptide at different loci), which are designated isoforms 1 -6 herein.
  • the Brassica plants described herein are homozygous or heterozygous for B. napus I MC 106RR QTL l and further comprise one or more mutant alleles at one or more fatB loci, wherein the mutant allele(s) at the one or more fatB loci results in the production of a
  • the Brassica plant contains one or more mutant alleles at two or more different fatB loci. In some embodiments, the Brassica plant contains one or more mutant alleles at three different fatB loci. In some embodiments, the Brassica plant contains mutant alleles at four different fatB loci.
  • the Brassica plant comprising a fatB mutation can have a nucleotide sequence encoding FATB isoform 1 , isoform 2, isoform 3, or isoform 4.
  • the plant can have a nucleotide sequence encoding isoforms 1 and 2; 1 and 3; 1 and 4; 2 and 3; 2 and 4; 3 and 4; 1 , 2, and 3; 1 , 2, and 4; 2, 3, and 4; or 1 , 2, 3, and 4;.
  • the Brassica plant can have a mutation in nucleotide sequences encoding FATB isoforms 1 , 2, and 3; 1 , 2, and 4; 2, 3, and 4; or 1 , 2, 3, and 4.
  • a mutation results in deletion of a 4HBT domain or a portion thereof of a FATB polypeptide.
  • the plants may be homozygous or heterozygous for the mutant alleles of each isoform.
  • FATB polypeptides typically contain a tandem repeat of the 4HBT domain, where the N-terminal 4HBT domain contains residues affecting substrate specificity (e.g. , two conserved methionines, a conserved lysine, a conserved valine, and a conserved serine) and the C-terminal 4HBT domain contains residues affecting catalytic activity (e.g. , a catalytic triad of a conserved asparagine, a conserved histidine, and a conserved cysteine) and substrate specificity (e.g. , a conserved tryptophan).
  • substrate specificity e.g. , two conserved methionines, a conserved lysine, a conserved valine, and a conserved serine
  • catalytic activity e.g. , a catalytic triad of a conserved asparagine, a conserved histidine, and a conserved cysteine
  • substrate specificity e
  • the mutation results in a non-conservative substitution of a residue in a 4HBT domain or a residue affecting substrate specificity.
  • the mutation is a splice site mutation.
  • the mutation is a nonsense mutation in which a premature stop codon (TGA, TAA, or TAG) is introduced, resulting in the production of a truncated polypeptide.
  • SEQ ID NOs: QTL5A.n, QTL5B.n, QTL5C.n and QTL5D.n set forth the nucleotide sequences encoding FATB isoforms 1 -4, respectively, and containing exemplary nonsense mutations that result in truncated FATB polypeptides.
  • SEQ ID NO: QTL5A.n is the nucleotide sequence of isoform 1 having a mutation at position 154, which changes the codon from CAG to TAG.
  • SEQ ID NO: QTL5B.n is the nucleotide sequence of isoform 2 having a mutation at position 695, which changes the codon from CAG to TAG.
  • QTL5C.n is the nucleotide sequence of isoform 3 having a mutation at position 276, which changes the codon from TGG to TGA.
  • SEQ I D NO: QTL5D.n is the nucleotide sequence of isoform 4 having a mutation at position 336, which changes the codon from TGG to TGA.
  • Brassica plants homozygous or heterozygous for QTL l of
  • IMC 106RR may also have decreased activity of FAD2, which is involved in the enzymatic conversion of oleic acid to linoleic acid, to confer an oleic acid content of about 70% to about 80% or about 80% or higher in the seed oil.
  • FAD2 The sequences for the wild-type fad2 genes from B. napus (termed the D form and the F form) are disclosed in W098/56239, which is incorporated herein by reference (SEQ ID NO: QTL4A.n, wherein nucleotide 3 16 is G, and SEQ ID NO: QTL4B.n, wherein nucleotide 515 is T, nucleotide 908 is G, and nucleotide 1001 is C).
  • the fad2 mutation is a G to A mutation at nucleotide 316 within the fad2-D gene (QTL4A, e.g., SEQ I D NO: QTL4A.n), which results in the substitution of a lysine residue for glutamic acid in a HECGH motif.
  • QTL4A e.g., SEQ I D NO: QTL4A.n
  • Such mutations are found within the variety IMC 129, which has been deposited with the ATCC under Accession No. 4081 1.
  • the fad2 mutation is a T to A mutation at nucleotide 51 5 of the fad2-F gene (e.g., SEQ ID
  • YLNNP motif amino acid 172 of the Fad2 F polypeptide
  • SEQ ID NO: QTL4B.p amino acid 172 of the Fad2 F polypeptide
  • the fad2 mutation is a G to A mutation at nucleotide 908 of the fad2-F gene (QTL4B), which results in the substitution of a glutamic acid for glycine in the
  • DRDYG1 LNKV motif amino acid 303 of the Fad2 F polypeptide.
  • Such a mutation is found within the variety Q4275, which has been deposited with the ATCC under Accession No. 97569. See U.S. Patent No. 6,342,658.
  • Another example of a suitable/cr ⁇ mutation can be the C to T mutation at nucleotide 1001 of the fad2-F gene (as numbered from the ATG) (SEQ ID ON: QTL4B.n, which results in the substitution of an isoleucine for threonine (amino acid 334 of the Fad2 F polypeptide).
  • Such a mutation is found within the high oleic acid variety Q741 5.
  • the modified fad2 loci comprise one or more nucleic acid sequence selected from the group consisting of SEQ ID NO: QTL4A.n wherein nucleotide 316 is A, SEQ ID NO: QTLB.n wherein nucleotide 515 is A, SEQ ID NO: QTL4B.n wherein nucleotide 908 is A, and SEQ ID NO: QTL4B.n wherein nucleotide 1001 is T.
  • the QTL4B locus may comprises a nucleic acid sequence of SEQ ID NO: QTL4B.n comprising one, two or three of the mutations selected from the group consisting of T to A mutation at nucleotide 51 5, G to A mutation at nucleotide 908, and C to T mutation at nucleotide 1001 .
  • the presence of one of the fad2-D (QTL4A) or fad2-F (QTL4B) mutations confers an oleic acid phenotype wherein the seed oil thereof has about 70% to about 80% oleic acid, while the presence of both fad2-D and fad2-F mutations confers an oleic acid phenotype wherein the seed oil thereof has more than about 80% oleic acid, even without QTL 1 mutation.
  • Q4275 contains the fad2-D mutation from IMC 129 and a fad2-F mutation at amino acid 303.
  • Q508 contains fad2-D mutation from IMC 129 and a fad2-F mutation at amino acid 1 72.
  • Q7415 contains the fad2-D mutation from IMC 129 and a fad2-F mutation at amino acid 334.
  • the presence of both fad2 mutations in Q4275, Q508, and Q7415 confers an oleic acid phenotype of greater than about 80% oleic acid.
  • Brassica plants also can exhibit reduced activity of delta- 1 5 desaturase (FAD3), which is involved in the enzymatic conversion of linoleic acid to a-linolenic acid.
  • FAD3 delta- 1 5 desaturase
  • the gene encoding FAD3 is referred to as fad3 in Brassica and Arabidopsis. Sequences of higher plant fad3 genes are disclosed in Yadav et al., Plant Physiol.. 103 :467-476 ( 1993), WO 93/1 1245, and Arondel et al., Science, 258: 1353- 1355 (1992). Decreased activity, including absence of detectable activity, of delta-15 desaturase can be achieved by mutagenesis.
  • Decreased activity can be inferred from the decreased level of linolenic acid (product) and in some cases, increased level of linoleic acid (the substrate) in the plant compared with a corresponding control plant (e.g. , the Brassica line Topas, ATCC deposit 40624).
  • parent plants can contain the mutation from the APOLLO or STELLAR B. napus variety that confers low linolenic acid.
  • the STELLAR and APOLLO varieties were developed at the University of Manitoba (Manitoba, Canada).
  • the parents contain the fad3A and/or fad3B mutation from IMC02 that confer a low linolenic acid phenotype.
  • IMC02 contains a mutation in both the fad3A and fad3B genes.
  • the mutation at the fad3 locus e.g., SEQ ID NO: QTL6A.n
  • the fad3A gene comprises a C to T mutation at position 2565, numbered from the ATG in genomic DNA, resulting in the substitution of a cysteine for arginine at position 275 of the encoded FAD3A polypeptide.
  • the mutation at the fad3 locus (e.g., SEQ I D NO: QTL6B.n) (fad3B gene) contains a G to A mutation at position 3053 from ATG in genomic DNA, located in the exon-intron splice site recognition sequence.
  • the mutation at the fad3 locus (e.g., SEQ ID NO: QTL6C.n) (fad3E gene) contains a G to A mutation at position 1756 from ATG in genomic DNA, located in the exon-intron splice site recognition sequence.
  • the mutation at the fad3 locus (e.g., SEQ ID NO: QTL6D.n) (fad3d gene) contains a 259 bp deletion that removes 165 bp from the l sl exon.
  • 1MC02 was obtained from a cross of IMCOl x Westar. See Example 3 of U.S. Patent No. 5,750,827.
  • IMCO l was deposited with the American Type Culture Collection (ATCC) under Accession No. 40579. IMC02 was deposited with the ATCC under Accession No. PTA- 6221.
  • Beta-ketoacyl-(acyl-carrier-protein) synthase III (kasIII)
  • Brassica plants also can exhibit reduced activity of KASII I or FabH ( ⁇ -ketoacyl-ACP synthase III), which is involved in the enzymatic synthesis of C 16 and C26 fatty acids.
  • the gene encoding FAD3 is referred to as kasIII in Brassica.
  • Decreased activity, including absence of detectable activity, of KASII I can be achieved by mutagenesis.
  • Decreased activity, including absence of detectable activity can be inferred from the decreased level of saturated fatty acids (e.g. C 16 and/or C26 fatty acids) in the plant compared with a corresponding control plant (e.g. the Brassica line Topas, ATCC deposit 40624).
  • the Brassica plant disclosed herein comprises nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to a modified kasIII locus (e.g., SEQ ID NO: QTL7.n).
  • QTL7 was identified on chromosome N 19 at position 1284751 24 the genomic sequence of B. napus line Salomon, ATCC deposit designation PTA- 1 1453, as described in WO 201 1 /075716, which is herein incorporated by reference.
  • the mutation identified in kasIII (SEQ ID NO: QTL7.n) relative to the
  • Australian cultivar Surpass 400 (Li et al consult 2003; wild type, SEQ ID NO: QTL7.n) is a transition from a "G” in the wild type to an "A” in Salomon at nucleotide 1250, SEQ ID NO: QTL7.n.
  • Brassica plants comprising the modified kasIII locus produces low amounts of saturated fatty acids in its seed oi l. More speci fically, the amino acid sequence encoded by the wi ld type comprises a glycine at position 252, whereas the KASI II of Salomon (i.e., encoded by the modified kasIII locus SEQ I D NO: QTL7.n) comprises a glutamic acid at that position (SEQ ID NO: QTL7.p).
  • the Brassica plants provided herein comprise one or more mutations at the QTL8 locus.
  • the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences within the segments defined by the chromosome N l (QTL8) SNP markers at positions 20772548 and 22780181 (e.g., between 20843387 and 21080816, or between 20874571 and 20979545) of the genomic sequence of B.
  • napus Salomon line ATCC deposit designation PTA- 1 1453, as described in WO 201 1 /075716, which is herein incorporated by reference, and wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
  • the Brassica plants comprise one, two, three, four, five, ten, fifteen or more QTL8 markers selected from the group consisting of:
  • the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences within the segments between any two chromosome N l (QTL8) SNP markers at positions selected from: 20772548, 20780679, 20843387, 208741 99, 20874571 , 20924967, 20979545, 21000713, 21057761 , 21080816, 21 126589, 21 1 75577, 212441 75, 21273898, 21301 953, 21 342623, 21 37881 5, 21425310, 21491979, 21 549878, 21597845, 21621627, 21648874, 21700869, 2174091 3, 21793927, 2182
  • the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences comprising any one or more (e.g., two, three, or four) chromosome Nl (QTL8) SNP markers at positions selected from: 20772548, 20780679, 20843387, 20874199, 20874571, 20924967, 20979545, 21000713, 21057761, 21080816, 21126589, 21175577, 21244175, 21273898, 21301953, 21342623, 21378815, 21425310, 21491979, 21549878, 21597845, 21621627, 21648874, 21700869, 217409
  • the nucleic acid comprises one or more sequences selected from the group consisting of SEQ ID NOs: 1-53, 1-54, 1-55, 1-56, 1-57, 1-58, 1-59, 1-60, 1-61, 1-62, 1-63, 1-64, 1-65, 1-66, 1-67, 1-68, 1-69, 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80, 1-81, 1-82, 1-83, 1-84, 1-85, 1-86, 1-87, 1-88, 1-89, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96, 1-97, 1-98, 1-99, 1-100, 1-101, 1-102, 1-103, and 1- 104.
  • the Brassica plants comprise a nucleic acid sequence having greater than 80% identity to all or part of the genomic sequences between the chromosome Nl (QTL8) SNP markers at positions 20772548 and 22780181 of the B. napus Salomon line, wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
  • the Brassica plants comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve single nucleotide polymorphisms on B. napus chromosome N 1 selected from the group consisting of C to T transitions identified in the B. napus Salomon line, ATCC deposit designation PTA-11453, at locations 20772548,
  • the Brassica plants comprise one, two, three, four, five, six, seven, or eight single nucleotide polymorphisms on B. napus chromosome Nl selected from the group consisting of C to T transitions at locations 20772548, 20780679, 20843387, 20874199, 20874571, 20924967, 20979545, and 21000713.
  • the Brassica plant comprises a B. napus N 1 chromosome having greater than 95%, 97.5%, 98%, 99%, 99.9%, 99.99%, 99.999% identity or having 100% identity to all or part of the genomic sequences of the B. napus Salomon line, ATCC deposit designation PTA- 1 1453, on an N l chromosomal segment selected from the group consisting of segments:
  • Table A B. napus position relative to the DH 12075 reference genome, wild-type allele, Salomon allele, flanking sequence and sequence ID number of SNPs identified in
  • the Brassica plants provided herein comprise one or more mutations at the QTL9 locus.
  • the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences within the segments defined by: the chromosome N 19 SNP markers at positions 1 1538807 and 18172630 (e.g., 12010676 and 13207412, 12378335 and 12979251 ) of the genomic sequence of B. napus line Salomon, ATCC deposit designation PTA-1 1453, wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
  • the Brassica plants comprise one, two, three, four, five, ten, fifteen or more QTL9 markers selected from the group consisting of:
  • the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences within the segments between any two chromosome Nl 9 (QTL9) SNP markers at positions selected from: 11538807, 11763228, 11855685, 12010676, 12205222, 12219881, 12355162, 12378335, 12507143, 12615691, 12847514, 12979251, 13003942, 13008581, 13207412, 13364132, 13429175, 13429687, 13460532, 13475876, 13504886, 13704881, 13925427, 14046125, 14135213, 14377562, 14776751, 14801661,
  • the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences comprising any one or more (e.g., two, three, or four) chromosome Nl 9 (QTL9) SNP markers at positions selected from: 11538807, 11763228, 11855685, 12010676, 12205222, 12219881, 12355162, 12378335, 12507143, 12615691, 12847514, 12979251, 13003942, 13008581, 13207412, 13364132, 13429175, 13429687, 13460532, 13475876, 13504886, 13704881, 13925427, 14046125, 14135213, 14
  • the nucleic acid comprises one or more sequences selected from the group consistingof SEQ IDNOs: 1-105, 1-106, 1-107, 1-108, 1-109, 1-110, 1-111, 1-112, 1-113, 1- 114, 1-115, 1-116, 1-117, 1-118, 1-119, 1-120, 1-121, 1-122, 1-123, 1-124, 1-125, 1-126, 1- 127, 1-128, 1-129, 1-130, 1-131, 1-132, 1-133, 1-134, 1-135, 1-136, 1-137, 1-138, 1-139, 1- 140, 1-141, 1-142, 1-143, 1-144, 1-145, 1-146, 1-147, 1-148, 1-149, 1-150, 1-151, 1-152, 1- 153, 1-154, 1-155, 1-156, 1-157, 1-158, 1-159, 1-160
  • the Brassica plants comprise a nucleic acid sequence having greater than 80% identity to r all or part of the genomic sequence between chromosome N 19 (QTL9) SNP markers at positions 1 1 538807 and 1 8172630 of the B. napus Salomon line, wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
  • the Brassica plants comprise comprising one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen single nucleotide polymorphisms on B. napus chromosome N 19 selected from the group consisting of C to T transitions identified in the B.
  • napus Salomon line ATCC deposit designation PTA- 1 1453, at locations 1 1 538807, 1 1 763228, 1 1 855685, 12010676, 12205222, 1221 9881 , 12355162, 12378335, 12507143, 1 261 5691 , 12847514, 12979251 , 13003942, 1 3207412, 13364132, 13429175, 13429687, 13460532, 13475876, 13504886, 13704881 , and a G to A transition at location 13008581 .
  • the Brassica plants comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, single nucleotide polymorphisms on B. napus chromosome N 1 9 selected from the group consisting of C to T transitions identified in the B. napus Salomon line, ATCC deposit designation PTA- 1 1453, at locations 1 1 538807, 1 1 763228, 1 1855685, 12010676, 12205222, 12219881 , 12355162, 12378335, 12507143, 1261 5691 , 12847514, 12979251 , 1 3003942, and a G to A transition at location 1 3008581 .
  • the Brassica plants comprise a B. napus N 19 chromosome having greater than 95%, 97.5%, 98%, 99%, 99.9%, 99.99%, 99.999% identity or having 100% identity to all or part of the genomic sequences of the B. napus Salomon line, ATCC deposit designation PTA- 1 1453, on an N 19 chromosomal segment selected from the group consisting of segments:
  • Table B B. napus position relative to the DH12075 reference genome, wild-type allele, Salomon allele, flanking sequence and sequence ID number of SNPs identified in
  • the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus line I MC 106RR and further comprising one or more mutant alleles at one or more fatB loci.
  • Two or more e.g. , three or four) different mutant alleles at one or more fatB loci may be combined in a plant by making a genetic cross between mutant lines.
  • a plant having a mutant allele at a fatB locus (QTL5A) encoding isoform 1 can be crossed or mated with a second plant having a mutant allele at a fatB locus (QTL5B) encoding isoform 2.
  • Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds can be screened in order to identify those seeds carrying both mutant alleles.
  • progeny are selected over multiple generations ⁇ e.g. , 2 to 5 generations) to obtain plants having mutant alleles at two different fatB loci.
  • a plant having mutant alleles at two or more different FATB isoforms can be crossed with a second plant having mutant alleles at two or more different fatB alleles, and progeny seeds can be screened to identify those seeds carrying mutant alleles at four or more different fatB loci.
  • progeny can be selected for multiple generations to obtain the desired plant.
  • the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus line IMC 106RR and further comprising one or more mutant al leles at one or more of the two fatA2 locus.
  • Mutant alleles at two different fatA2 locus may be combined in a plant by making a genetic cross between mutant lines, similar to the method described supra.
  • the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus line IMC 106RR and further comprising one or more mutant alleles at one or more of the four fad2 loci.
  • Mutant alleles at two different fad2 loci may be combined in a plant by making a genetic cross between mutant lines, simi lar to the method described supra.
  • the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus line 1MC 106RR and further comprising one or more mutant alleles at three or more fad3 loci.
  • Mutant alleles at three or four different fad3 loci may be combined in a plant by making a genetic cross between mutant lines, simi lar to the method described supra.
  • the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus line 1MC 106RR and further comprising one or more mutant alleles at one fatA2 locus (QTL3A), and one or more mutant alleles at one, two or more ⁇ e.g. , three, or four) of the four different fatB loci (QTL5A, QTL5B, QTL5C, and QTL5D).
  • a plant having a mutant allele at a fatA2 locus can be crossed or mated with a second plant having mutant alleles at two or more different fatB loci.
  • progeny seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds can be screened in order to identify those seeds carrying mutant fatA2 and fatB alleles. Progeny can be selected over multiple generations (e.g. , 2 to 5 generations) to obtain plants having a mutant allele at a fatA2 locus and mutant alleles at two or more different fatB loci. As described herein, plants having a mutant al lele at a fatA2b locus and mutant alleles at three or four different fatB loci have a low total saturated fatty acid content that is stable over different growing conditions, i.e. , is less subject to variation due to warmer or colder temperatures during the growing season.
  • plants having mutations in fatA2 and fatB loci may exhibit a substantial reduction in amounts of both 16:0 and 1 8:0 in seed oil.
  • the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus line 1MC 106RR and further comprising one or more mutant alleles at one or more mutant alleles at one, two or more ⁇ e.g., three or four) of the four different/o/5 loci (QTL5A, QTL5B, QTL5C, and QTL5D).
  • the Brassica plants described herein that are homozygous or heterozygous for the QTL1 of the B. napus I C 106RR and further comprise one or more mutant alleles at one or more mutant alleles at one fatA2 locus, and one or more mutant alleles at one, two, three, or four of the four different fatB loci.
  • FAD2 in combination with reduced activity of FATA2 and/or FATB.
  • the Brassica plants described herein are homozygous or heterozygous for IMC 106RR QTL l and further comprise one or more mutant alleles at one or more (e.g. two, three, or four) of the four different fad2 locus (e.g. , QTL4A and QTL4B) and one or more mutant alleles at:
  • one fatA2 locus e.g. , QTL3A
  • one, two or more e.g., three or four
  • QTL5A, QTL5B, QTL5C, and QTL5D the four different fatB loci
  • Plants comprising QTLl and exhibit reduced activity of FAD3, and one, two or three enzymes selected from FATA2, and FATB; and/ or further exhibit reduced activity of FAD2.
  • the Brassica plants are homozygous or heterozygous for IMC 106RR QTL l and further comprise one or more mutant alleles at three, four, five, or six of the six different fad3 loci (QTL6A, QTL6B, QTL6C, and QTL6D), and one or more mutant alleles at:
  • one fatA2 locus e.g. , QTL3 A
  • one, two or more e.g., three or four
  • QTL5A, QTL5B, QTL5C, and QTL5D the four different/o/5 loci
  • the Brassica plants homozygous or heterozygous for IMC 106RR QTL l and further comprise one or more mutant alleles at three, or four of the four different fad3 loci (QTL6A, QTL6B, QTL6C, and QTL6D), one or more (e.g. two, three, or four) of the four different/oi/2 loci (e.g. , QTL4A and QTL4B), and:
  • one fatA2 locus e.g. , QTL3Aand/or one, two or more (e.g., three or four) of the four different/a/5 loci (QTL5A, QTL5B, QTL5C, and QTL5D).
  • Plants comprising QTLl and (1) QTL7 and/or QTL8 and/or QTL9, and/or further exhibit reduced activity of (2) one, two or three enzymes selected from FATA2, and FATB, and/ or (3) FAD2 and/or FAD3
  • Brassica plants that are homozygous or heterozygous for QTL l of the B. napus line IMC 106RR also comprise one or more mutant alleles at the locus on a kasIII locus (QTL 7) and/or chromosome N 1 (QTL8) and/or the locus on the chromosome N 19 (QTL9), as described supra.
  • Brassica plants that are homozygous or heterozygous for QTL 1 of the B. napus line IMC 106RR also comprise one or more mutant alleles at:
  • any of the plants described herein that are homozygous or heterozygous for QTL 1 of IMC 106RR may be homozygous or heterozygous for one or more mutant alleles at one, two, three, four, five, six, seven, eight, nine, or ten different loci selected from the group consisting of, QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL7, QTL8, and QTL9.
  • Brassica plants comprising QTL 1 further comprise ( 1 ) one or more mutant alleles at three or four of different loci selected from the group consisting of QTL6A, QTL6B, QTL6C, and QTL6D; and/or (2) one or more modified ⁇ e.g. , mutant) alleles at one, two, three, four, five, six, seven, eight, nine or ten different loci selected from the group consisting of QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL7, QTL8, and QTL9.
  • mutant alleles at loci QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL7, QTL8, and QTL9 are the alleles recited in Table C.
  • the plants described herein may be modified and/or selected to display a herbicide tolerance trait. That trait can be introduced by selection with the herbicide for which tolerance is sought, or by transgenic means where the genetic basis for the tolerance has been identified. Accordingly, the plants described herein, or parts thereof such as cel ls or protoplasts, may display tolerance to a herbicide selected from the group consisting of imidazolinone, dicamba, cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, L-phosphinothricin, triazine and benzonitrile. Where the plants have been genetically modified to acquire herbicide tolerance by transgenic means they may be non- transgenic to the extent of all other traits except herbicide tolerance. 7.0 Production of Hybrid Brassica Varieties
  • Hybrid Brassica varieties can be produced by preventing self-pollination of female parent plants ⁇ i.e., seed parents), permitting pollen from male parent plants to fertilize such female parent plants, and allowing F
  • Self- pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development.
  • pollen formation can be prevented on the female parent plants using a form of male steri lity.
  • male sterility can be cytoplasmic male sterility (CMS), nuclear male sterility, molecular male sterility wherein a transgene inhibits microsporogenesis and/or pollen formation, or be produced by self-incompatibility.
  • CMS cytoplasmic male sterility
  • nuclear male sterility nuclear male sterility
  • molecular male sterility wherein a transgene inhibits microsporogenesis and/or pollen formation, or be produced by self-incompatibility.
  • Female parent plants containing CMS are particularly useful
  • CMS can be, for example, of the ogu (Ogura), ⁇ , ⁇ , tour, or mur type. See, for example, Pellan-Delourme and Renard, 1987, and Pellan-Delourme and Renard, 1988. See Riungu and McVetty, 2003 for a description of ⁇ , ⁇ , tour, and mur type CMS.
  • the male parent plants typically contain a fertility restorer gene to ensure that the F
  • a fertility restorer gene that can overcome the Ogura type CMS.
  • Non-limiting examples of such fertility restorer genes include the Kosena type fertility restorer gene (U.S. Patent No. 5,644,066) and Ogura fertility restorer genes (U.S. Patent Nos. 6,229,072 and 6,392, 127).
  • male parents can be used that do not contain a fertility restorer.
  • hybrids produced from such parents are male sterile. Male sterile hybrid seed can be inter-planted with male fertile seed to provide pollen for seed-set on the resulting male sterile plants.
  • the methods described herein can be used to form single-cross Brassica F
  • the parent plants can be grown as substantially homogeneous adjoining populations to facilitate natural cross-pollination from the male parent plants to the female parent plants.
  • the Fi seed formed on the female parent plants is selectively harvested by conventional means.
  • One also can grow the two parent plants in bulk and harvest a blend of F
  • three-way crosses can be carried out wherein a single-cross F
  • the overall oleic acid content of the vegetable oil may be reduced over that of a single-cross hybrid; however, the seed yield will be further enhanced in view of the good agronomic performance of both parents when making the second cross.
  • double-cross hybrids can be created wherein the Fi progeny of two different single-crosses are themselves crossed. Self-incompatibility can be used to particular advantage to prevent self-pollination of female parents when forming a double-cross hybrid.
  • Hybrids described herein have good agronomic properties and exhibit hybrid vigor, which results in seed yields that exceed that of either parent used in the formation of the Fi hybrid.
  • yield can be at least 10% (e.g., 10% to about 20%, 10% to about 1 5%, about 1 5% to about 20%, or about 25% to about 35%) above that of either one or both parents.
  • the yield exceeds that of open-pollinated spring canola varieties such as 46A65 (Pioneer) or Q2 (University of Alberta), when grown under similar growing conditions.
  • yield can be at least 10% (e.g. , 10% to about 1 5% or about 15% to about 20%) above that of an open-pollinated variety.
  • Hybrids described herein typically produce seed oil having very low levels of glucosinolates (less than30 ⁇ l/gram of de-fatted meal at a moisture content of 8.5%).
  • hybrids can produce seed oil having less than 20 ⁇ of glucosinolates/gram of de-fatted meal.
  • hybrids can incorporate mutations that confer low glucosinolate levels. See, for example, U.S. Patent No. 5,866,762.
  • Glucosinolate levels can be determined in accordance with known techniques, including high performance liquid chromatography (HPLC), as described in ISO 9167- 1 : 1992(E), for quantification of total, intact
  • TMS trimethy Isilyl
  • Brassica plants disclosed herein are useful for producing canola oils with low or no total saturated fatty acids.
  • seed oil of Brassica plants described herein may have a total saturated fatty acid content of about 2.5% to about 5.5%, about 3% to about 5%, about 3% to about 4.5%, about 3.25% to about 3.75%, about 3% to about 3.5%, about 3.4% to about 3.7%, about 3.6% to about 5%, about 4% to about 5.5%, about 4% to about 5%, or about 4.25% to about 5.25%.
  • an oil has a total saturated fatty acid content of about 4% to about 5.5%, an oleic acid content of about 60% to about 70% (e.g.
  • an oil has a total saturated fatty acid content of about 2.5% to about 5.5% (e.g. , about 4% to about 5%), an oleic acid content of about 71 % to about 80% (e.g.
  • a-linolenic acid content of about 2% to about 5.0% (e.g., about 2% to about 2.8%, about 2.25% to about 3%, about 2.5% to about 3%, about 3% to about 3.5%, about 3.25% to about 3.75%, about 3.5% to about 4%, about 3.75% to about 4.25%, about 4% to about 4.5%, about 4.25% to about 4.75%, about 4.5% to about 5%).
  • a canola oil can have a total saturated fatty acid content of about 2.5% to about 5.5%, an oleic acid content of about 78% to about 80%, and an a-linolenic acid content of no more than about 4% (e.g. , about 2% to about 4%).
  • an oil has a total saturated fatty acid content of about 3.5% to about 5.5% (e.g., about 4% to about 5%), an oleic acid content of about 81 % to about 90% (e.g. , about 82% to about 88% or about 83% to about 87% oleic acid) and an a-linolenic acid content of about 2% to about 5% (e.g. , about 2% to about 3% or about 3% to about 5%).
  • an oil has a total saturated fatty acid content of no more than about
  • Seed oil of Brassica plants described herein can have a palmitic acid content of about 3.0% to about 5.5% (e.g., about 3.6% to about 5.2%).
  • the stearic acid content of such oils can be about 1.0% to about 3.5% (e.g. , about 1 .3% to about 3.0%).
  • the arachidic acid content of the oil can be about 0.5% to about 1.5% (e.g., about 0.6% to about 1 .1 %).
  • the docosanoic acid content of the oil can be about 0.3% to about 0.8% (e.g. , about 0.4% to about 0.65%).
  • Seed oil of Brassica plants described herein can have an eicosenoic acid content greater than about 1 .6%, e.g. , about 1 .6% to about 1.9%, about 1 .7% to about 2.3%, about
  • Seed oil of Brassica plants described herein can have a linoleic acid content of about 3% to about 20%, e.g. , about 3.4% to about 5%, about 3.75% to about 5%, about 8% to about 10%, about 10% to about 12%, about 1 1 % to about 1 3%, about 1 3% to about 16%, or about 14% to about 18%, in addition to a low total saturates content.
  • Seed oil of Brassica plants described herein have an erucic acid content of less than about 2% (e.g., less than about 1 %, about 0.5%, about 0.2%, or about 0.1 %) in addition to a low total saturates content.
  • the fatty acid composition of seed oil of Brassica plants can be determined by first crushing and extracting oil from seed samples (e.g., bulk seed samples of 10 or more seeds). TAGs in the seed are hydrolyzed to produce free fatty acids, which then can be converted to fatty acid methyl esters and analyzed using techniques known to the ski lled artisan, e.g. , gas- liquid chromatography (GLC) according to AOCS Procedure Ce l e-91 .
  • GLC gas- liquid chromatography
  • NI R Near infrared
  • Seeds harvested from plants described herein can be used to make a crude canola oil or a refined, bleached, and deodorized (RBD) canola oil with a low or no total saturated fatty acid content.
  • Harvested canola seed can be crushed by techniques known in the art.
  • the seed can be tempered by spraying the seed with water to raise the moisture to, for example, about 8.5%.
  • the tempered seed can be flaked using a smooth roller with, for example, a gap setting of 0.23 to 0.27 mm. Heat may be applied to the flakes to deactivate enzymes, facilitate further cell rupturing, coalesce the oil droplets, or agglomerate protein particles in order to ease the extraction process.
  • oil is removed from the heated canola flakes by a screw press to press out a major fraction of the oil from the flakes. The resulting press cake contains some residual oil.
  • Crude oil produced from the pressing operation typically is passed through a settling tank with a slotted wire drainage top to remove the solids expressed out with the oil in the screw pressing operation.
  • the clarified oil can be passed through a plate and frame fi lter to remove the remaining fine solid particles.
  • Canola press cake produced from the screw pressing operation can be extracted with commercial n-Hexane. The canola oil recovered from the extraction process is combined with the clarified oil from the screw pressing operation, resulting in a blended crude oil.
  • Free fatty acids and gums typically are removed from the crude oil by adding food grade phosphoric acid and heating the acidified oil in a batch refining tank.
  • the acid serves to convert the non-hydratable phosphatides to a hydratable form, and to chelate minor metals that are present in the crude oil.
  • the phosphatides and the metal salts are removed from the oil along with the soapstock.
  • the oi l-acid mixture is subsequently treated with sodium hydroxide solution to neutral ize the free fatty acids and the remaining phosphoric acid in the acid-oil mixture.
  • the neutralized free fatty acids, phosphatides, etc. (soapstock) are drained off from the neutralized oil.
  • a water wash may be done to further reduce the soap content of the oil.
  • the oil may be bleached and deodorized before use, if desired, by techniques known in the art.
  • Oils obtained from the Brassica plant described herein can have increased oxidative stability, which can be measured using, for example, an Oxidative Stability Index Instrument (e.g. , from Omnion, Inc., Rockland, MA) according to AOCS Official Method Cd 12b-92 (revised 1993). Oxidative stability is often expressed in terms of "AOM" hours.
  • Oxidative Stability Index Instrument e.g. , from Omnion, Inc., Rockland, MA
  • Seeds were analyzed for fatty acid profile using gas chromatography. Seeds were crushed, and lipids were extracted using an alkaline extraction method (potassium hydroxide, sodium chloride, iso-octane). The sample was centrifuged and the top layer was used for GC analysis. LSMEANS for each DH line were determined using the GLM procedure in the SAS software package (SAS Institute, 2004).
  • Genotyping was done using the lllumina (San Diego, CA) Brassica 60K Infinium array at DNA Landmarks (Quebec, Canada). The final list of 1 , 1 79 markers used in linkage map construction was selected based on GenTrain genotype scores above 0.75 as suggested by lllumina followed by selection for those which lack an inter-homoeologous polymorphism (Trick et al., 2009).
  • the genetic linkage map was constructed in JoinMap3 (Van Ooijen and Voorrips, 2001 ) using a threshold recombination frequency of ⁇ 0.25 and a minimum logarithm of the odds ratio (LOD) score of 6 for grouping loci into linkage groups.
  • LOD odds ratio
  • the osambi function ( osambi, 1944) was used to calculate genetic distances. Each linkage group was named based on the nomenclature recommended by the Multinational Brassica Genome Project steering committee (http://www.brassica.info/resource/maps/Ig- assignments.php). The map was analyzed further in the R qtl program of the R statistical package (Broman et al., 2003; Broman and Sen, 2009) to confirm marker orders and assess general map quality.
  • QTL mapping was performed using Haley-Knott Regression (Haley and Knott, 1992) in R/qtl using 1 cM steps. QTL were selected based on significance thresholds made from 1000 permutations (Churchill and Doerge, 1994). Secondary analysis of all polymorphic markers present within the QTL region defined during genome-wide scans were conducted by regressing fatty acid mean values onto molecular marker genotypes (Whittaker, Thompson and Visscher, 1996). For this analysis, markers were aligned in the order of their estimated physical locations.
  • DNA libraries of each parent were sequenced on one lane of an Illumina HiSeq 2000 (San Diego, CA) sequencer to generate 2 x 100 paired-end reads.
  • Genome-wide QTL scans discovered one major locus on chromosome N 1 5 that explained a significant proportion of the variation in C 1 8:0, C20:0, C22:0 and total saturates (Table 1 ).
  • Table 1 Summary of QTL location, logarithm of odds (LOD) score, LOD threshold*, percent variance explained (R 2 ), trait mean of lines carrying I C 106RR allele, trait mean of lines carrying Wichita allele and mean differences among lines carrying parental alleles.
  • DH lines carrying the IMC 106RR allele were significantly lower in each fatty acid component than DH lines carrying the Wichita allele.
  • Further analysis of the QTL region using the single marker regression approach found a distinct peak in the R 2 value of the marker located at 42,877,3 1 8 bp and a significant improvement in the R 2 value relative to the marker located at 42,605, 1 57 bp identified in the original genome-wide scan as the most highly correlated (Table 2).
  • the peak at this location is considered the "center” of the QTL from which we define the QTL interval to encompass a 1 Mb region on either side.
  • the physical locations listed in Table 2 are based on a C genome assembly.
  • the QTL interval is defined as spanning from 43,730,765 - 45,730,765 bp on N l 5 of the DH 12075 B. napus genome assembly.
  • SNP discovery within the defined QTL interval was performed as described. A list of 38 SNPs was generated (Table 4) requiring all selected SNPs to pass all quality thresholds (detailed above), to be homozygous variant and to be unique to the IMC 106RR genotype (i.e., different than the Wichita sequence and the DH 12075 reference sequence).
  • QTL3A and QTL7 QTL3A and QTL4A, QTL3A and QTL4B, QTL3A and QTL5A, QTL3A and QTL5B, QTL3A and QTL5C, QTL3A and QTL5D, QTL3A and QTL8, QTL3A and QTL9,
  • QTL4A and QTL4B QTL4A and QTL5A, QTL4A and QTL5B, QTL4A and QTL5C, QTL4A and QTL5D, QTL4A and QTL8, QTL4A and QTL9,
  • QTL4B and QTL5A QTL4B and QTL5B, QTL4B and QTL5C, QTL4B and QTL5D, QTL4B and QTL8, QTL4B and QTL9,
  • QTL5A and QTL5B QTL5A and QTL5C, QTL5A and QTL5D, QTL5A and QTL8, QTL5A and QTL9,
  • QTL5B and QTL5C QTL5B and QTL5D, QTL5B and QTL8, QTL5B and QTL9,
  • QTL3A, QTL7, QTL4A and QTL4B QTL3A, QTL7, QTL4A and QTL5A, QTL3A, QTL7, QTL4A and QTL5B, QTL3A, QTL7, QTL4A and QTL5C, QTL3A, QTL7, QTL4A and QTL5D, QTL3A, QTL7, QTL4A and QTL8, QTL3A, QTL7, QTL4A and QTL9, QTL3A, QTL7, QTL4B and QTL5A, QTL3A, QTL7, QTL4B and QTL5B, QTL3A, QTL7, QTL4B and QTL5C, QTL3A, QTL7, QTL4B and QTL5D, QTL3A, QTL7, QTL4B and QTL8, QTL3A, QTL7, QTL4B and QTL9, QTL3A, QTL7, QTL7, Q
  • QTL3A, QTL4A, QTL5B, QTL5D and QTL8 QTL3A, QTL4A, QTL5B, QTL5D and QTL9
  • QTL3A, QTL4B, QTL5A, QTL5C, QTL5D and QTL8 QTL3A, QTL4B, QTL5A,QTL5C, QTL5D and QTL9,
  • QTL4B QTL5A, QTL5B,QTL5C, QTL5D and QTL9, QTL4B, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,

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Description

NOVEL QTL OF BRASSICA PLANT CORRELATED TO FATTY ACID PROFILE
PRIORITY CLAIM
This application claims priority to U.S. Provisional Application No. 62/086, 165. Filed December 1 , 2014, the contents of which is incorporated herein by reference in its entirety, including drawings.
TECHNICAL FIELD
This disclosure relates to Brassica plants and, more particularly, Brassica plants having one or more mutations at a novel quantitative trait locus denoted "QTL 1 " that correlates to the fatty acid profile of their seed oil. The Brassica plants may also contain a modified fatty acyl-acyl carrier protein thioesterase A2 (fatAI) locus (e.g. , QTL3A), one or more modified fatty acid desaturase (fad2) loci (e.g. , QTL4A,and QTL4B), one or more modified fatty acyl-acyl carrier protein thioesterase B (fatB) loci (e.g. , QTL5A, QTL5B, QTL5C, QTL5D), a modified beta-ketoacyl-(acyl-carrier-protein) synthase I I I (kasIII) locus (e.g., QTL 7), a modified locus on the chromosome N l (QTL8), and/or a modified locus on the chromosome N 19 (QTL9) which may further contribute to a desired fatty acid phenoty pe (e.g., total saturated fatty acid content of about 8.5% or less in combination with an oleic acid content of about 62% to about 85%, a linoleic acid content of about 8% to about 10%, and/or an a-linolenic acid content of no more than about 4%). The Brassica plants may further comprise three, or four modified fad3 loci (e.g. , QTL6A, QTL6B, QTL6C, and QTL6D). The sequences for this application are shown in Table D.
BACKGROUND
In recent years, diets high in saturated fats have been associated with increased levels of cholesterol and increased risk of coronary heart disease. As such, current dietary guidelines indicate that saturated fat intake should be no more than 10 percent of total calories. Based on a 2,000-calorie-a-day diet, this is about 20 grams of saturated fat a day. While canola oil typically contains only about 7% to about 8% saturated fatty acids, a decrease in its saturated fatty acid content would improve the nutritional profile of the oil.
Most of the traits we observe in nature are under the control of many genes and, due to genetic variation within those genes, have a large distribution of trait values within a population. These traits have been termed quantitative traits and the loci controlling them are known as QTLs. QTLs are regions of the genome which explain variation in continuously distributed phenotypes, and almost always contain many (hundreds or more) genes (Mackay, 2001 ).
Construction of a genetic map can be accomplished by analyzing the segregation of alleles in a recombinant population (Haldane, 1919). A recombinant population of plants is often generated from a directed cross between two inbred lines, hereafter known as the parents. Recombination during meiosis results in a population of individuals whose genotypes are a mosaic of the parental alleles. Parental alleles can be tracked via a number of marker methods including morphology (Sturtevant, 1913; Sax, 1923), isozyme (Hunter and Markert, 1957) and DNA (Botstein et al., 1980; Nakamura et al., 1987). A DNA marker is simply a DNA sequence that shows sequence polymorphism among individuals within a species (Andersen and Lubberstedt, 2003). Analysis of the cosegregation of marker polymorphisms (alleles) within the recombinant population allows a researcher to construct a map of the estimated locations of each allele relative to others. Marker alleles that are found together more often are assumed to be located closer together because of a limitation in crossover events due to physical linkage (Muller, 1 916). Pairwise comparisons of the frequency of each allelic combination are used to create a genetic map which provides a visual illustration of their location on an organism's chromosomes.
QTL mapping is essentially the association of a locus with variation in a quantitative phenotype (Mauricio, 2001 ). Two pieces of information are needed for a QTL mapping experiment. First, genotype data for each individual within the recombinant population at each marker locus is recorded as the parental allele from which it originates. Second, phenotype data for the target trait is measured for each individual of the recombinant population. Statistical association analyses of the phenotype data and the marker data will reveal loci which explain a significant proportion of the variation in the phenotype data. The association between genetic map positions and a phenotype can then be inferred. The final product is a map of those areas of the genome containing QTLs.
SUMMARY
Provided herein are plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), comprising one or more mutations in the QTL 1 locus, a novel quantitative trait locus identified in a doubled haploid (DH) population of 225 lines derived from a cross between I MC 106RR (Cargill, Incorporated, National Registration No. 5 1 1 8) and the biennial variety Wichita (Rife et al., 2001 ; Registration No. CV- 1 9, PI 612846). Also provided herein are methods of generating such plants and seeds, oils derived from such plants and seeds, and methods of making these oils.
The QTL 1 locus is defined by its correlation to variations in the fatty acid profi le (e.g., C 1 8:0, C20:0, C22:0, and/or total saturates) of seed oil from the DH cross lines, and by the SNP markers identified herein. The QTL 1 locus is believed to reside upon Brassica napus chromosome 15. However, it is understood that the locus is defined not by chromosomal position but by its SNP markers and its contribution to the fatty acid content of seed oil, and therefore the locus may appear on other chromosomes, particularly in progeny.
In certain embodiments, seeds of plants comprising one or more mutations in QTL 1 may yield oil having a total saturated fatty acid content of about 8.5% or less (e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5%). The palmitic acid content of the oil may be about 3.0% to about 5.5% (e.g. , about 3.0% to about 5.5%, about 3.0% to about 5%, or about 3.5% to about 4.5%). The stearic acid content of the oil may be about 1 .0% to about 3.5% (e.g. , about 1.0% to about 3.0%, about 1.0% to about 2.0%, or about 1.3% to about 2.0%) The arachidic acid (i.e. , eicosanoic acid) content of the oil may be about 0.5% to about 1.5% (e.g. , about 0.5% to about 1 .2%, about 0.5% to about 1 .0%, or about 0.6% to about 1.0%). The docosanoic acid content of the oil may be about 0.3% to about 0.8% (e.g. , about 0.3% to about 0.7%, about 0.4% to about 0.7%, or about 0.4% to about 0.65%). In addition, the oil may have an oleic acid content of about 62% to about 85% or higher (e.g. , about 62% to about 65%, about 65% to about 72%, about 72% to about 75%, about 75% to about 80%, about 80% to about 84% or about 82% to about 85%), a linoleic acid content of about 8% to about 10%, and/or an a-linolenic acid content of no more than about 4% (e.g., about 2% to about 4%).
In certain embodiments, the plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein comprising one or more mutations in QTL 1 (N 1 5) may further comprise ( 1 ) one or more QTLs and/or known genetic variants (mutations) selected from the group consisting of: falA2 mutations (e.g. , QTL3A),fad2 mutations (e.g. , QTL4A and QTL4B),/a/5 mutations (e.g., QTL5A, QTL5B, QTL5C, and QTL5D), kasIII mutations (e.g. , QTL7), mutations on the chromosome N l (e.g. , QTL8), and mutations on the chromosome N 19 (e.g. , QTL9); and/or (2) one or more mutant alleles at three or four fad3 loci (e.g. , QTL6A, QTL6B, QTL6C, and QTL6D). In certain embodiments, plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein comprising one or more mutations in QTL1 may further comprise one or more mutant alleles at:
(i) one or more fatA2 loci and/or fatB loci.
Alternatively or in addition to these mutant alleles, plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein may further comprise one or more mutant al leles at:
(ii) three or four fad3 loci; and/or
(iii) one or more fad2 loci.
In certain embodiments, plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein comprising one or more mutations in QTL1 locus may further comprise one or more mutant alleles at:
(i) one or more fatB loci and/or fad2 loci.
Alternatively or in addition to these mutant alleles, plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein may further comprise one or more mutant alleles at:
(ii) three or four fad3 loci.
In certain embodiments, plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein comprising one or more mutations in QTL1 and one or more of fatA2 mutations, one or more fatB mutations, and/or one or more mutations in QTL7 and/or QTL8 and/or QTL9 as described above may produce oils with a total saturated fatty acid content less than about 8.5% (e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5% total saturates), oils having less than about 3.6% saturated fatty acids (e.g. , about 2.0% to about 3.6%, about 2.5% to about 3.6% or about 3% to about 3.6% total saturates), oils having palmitic acid (C 16:0) content of about 6.0% or less, oils having stearic acid (C 18:0) content of about 4.0% or less, oils having arachidic acid (C20:0) content of about 1.5% or less, and/or oils having docosanoic acid (C22:0) content of about 0.8% or less.
In certain embodiments, plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), provided herein comprising one or more mutations in
QTL 1 and one or more mutations at one or more loci selected from the group consisting of fatA2 locus (e.g. , QTL3A),fatB loci (e.g., QTL5A, QTL5B, QTL5C, and QTL5D), kasIII locus (e.g. , QTL7), loci on the chromosome 1 (e.g. , QTL8), and loci on the chromosome N 19 (e.g., QTL9) may further comprise mutations at one or more fad2 alleles (e.g. , QTL4A and QT43B) and/or three or more fad3 alleles (e.g. , QTL6A, QTL6B, QTL6C, and QTL6D). In certain embodiments, the presence or absence of specific combinations of mutations at fad2 and/or fad3 alleles may be used to tailor the oleic acid and a-linolenic acid content of oils produced by the plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), to the desired end use of the oil.
In certain embodiments, the present disclosure provides Brassica plants (e.g. , Brassica napus, Brassica juncea, Brassica rapa, Brassica oleracea, or Brassica carinata), including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), that comprise one or more mutations in QTL 1 on chromosomes N l 5 relative to the wild-type allele in
DH 12075. Plants comprising mutations in QTL 1 can be derived from IMC 106RR either as the result of a cross in which IMC 106RR is directly involved, or by using plants derived from IMC 106RR comprising all or part of the genome corresponding to QTL 1 . Plants bearing the region of IMC 106RR corresponding to QTL1 can be DH plant(s) derived from a cross between IMC106RR, Fi hybrids of IMC 106RR, or F2, F3, F4 or subsequent generations of progeny derived from IMC 106RR. Such plants may further comprise one or more mutations at one, two, three, four, five, six, seven, eight, nine, or ten selected from the group consisting of QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL7, QTL8, and QTL9. In another embodiment, Brassica plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), comprising one or more mutations in QTL 1 further comprise ( 1 ) one or more mutant alleles at three, or four, of different loci selected from the group consisting of QTL6A, QTL6B, QTL6C, and QTL6D; and/or (2) one or more modified (e.g. , mutant) alleles at one, two, three, four, five, six, seven, eight, nine or ten loci selected from the group consisting of QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL7, QTL8, and QTL9. In certain embodiments, the mutant alleles at loci QTL3A,
QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D,QTL 7, QTL8, and QTL9 are the alleles recited in Table C.
In certain embodiments, methods are provided herein for producing oil from the Brassica plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), disclosed herein. In certain embodiments, these methods comprise ( 1 ) crushing seeds produced from at least one Brassica plant comprising one or more mutations in QTL1 that contributes to the reduced saturated fatty acid content in IMC 106RR described herein, and one or more mutations selected from the group consisting of fatA2 mutations (e.g. , QTL3A), fad2 mutations (e.g. , QTL4A and QTL4B),/a/5 mutations (e.g., QTL5A, QTL5B, QTL5C, and QTL5D), kasIII mutations (e.g. , QTL7), mutations on the chromosome N l (e.g. , QTL8), and mutations on the chromosome N l 9 (e.g. , QTL9); and (2) extracting the oil from the crushed seeds, where the oil after refining, bleaching, and deodorizing, has a total saturated acid content of about 2.5% to about 8.5%. The oil further may further comprise about 1 .6% to about 2.3% eicosenoic acid, about 78% to about 80% oleic acid, about 8% to about 10% linoleic acid, and/or about 2% to about 4% a-linolenic acid. Brassica plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), used in these methods may further comprise three, or four, fad3 mutations (e.g. , QTL6A, QTL6B, QTL6C, and QTL6D).
Also provided herein in certain embodiments are methods for making a Brassica plant comprising one or more mutations in QTL1 of chromosome N l 5 that contributes to the total saturated fatty acid content of about 8.5% or less (e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5% total saturates) in IMC 106RR seed oil and one or more mutations at one, two, three, four, five, six, seven, eight, nine or ten different loci selected from the group consisting of fatA2 locus (e.g. , QTL3A),fad2 loci (e.g. , QTL4A andQTL4B),/a/5 loci (e.g., QTL5A, QTL5B, QTL5C, and QTL5D), kasIII locus (e.g., QTL7), locus on the chromosome N l (e.g. , QTL8), and locus on the chromosome N 19 (e.g. , QTL9). In certain embodiments, these methods comprise:
a) crossing one or more first Brassica parent plants bearing a mutation in QTL 1 that contributes to the reduced saturated fatty acid content in IMC 106RR with one or more second Brassica parent plants,
wherein said first Brassica parent plants comprise a nucleic acid sequence having greater than 80% identity (e.g. , 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.8%, 99.9% or 99.99%) to all or part of the genomic sequences between the chromosome N l 5 (QTL 1 ) SNP markers at positions 43730765 and 45730765 of the B. napus line IMC 1066RR, wherein said part of the genomic sequences of the B. napus IMC 106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides, wherein said first parent plant optionally comprises one or more mutant alleles at one, two, three, four or more different loci selected from the group consisting offatA2 locus (e.g. , QTL3A), fad2 loci (e.g. , QTL4A and QTL4B), fatB loci (e.g., QTL5A, QTL5B, QTL5C, and QTL5D), kasIII locus (e.g. , QTL7), locus on the chromosome N 1 (e.g. , QTL8), and locus on the chromosome N 19 (e.g. , QTL9), wherein said one or more second Brassica parent plants optionally comprise a mutant allele at one, two, three, four or more different loci selected from the group consisting of fatA2 locus (e.g., QTL3A),fa 2 loci (e.g. , QTL4A and QTL4B),fatB loci (e.g., QTL5A, QTL5B, QTL5C, and QTL5D), kasIII locus (e.g., QTL7), locus on the chromosome N l (e.g. , QTL8), and locus on the chromosome N l 9 (e.g. , QTL9) of said first Brassica parent;
and
b) selecting, for one, two, three, four, or five or more generations, for progeny plants having:
(i) all or part of the genomic sequences between the chromosome 15 (QTL 1 ) SNP markers at positions 43730765 and 45730765, wherein said part of the genomic sequences of the B. napus IMC 106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides,
and
(ii) said mutant alleles at one, two, three, four or more different loci selected from the group consisting of fatA2 locus (e.g. , QTL3 A), fad2 loci (e.g. , QTL4A and QTL4B), fatB loci (e.g., QTL5A, QTL5B, QTL5C, and QTL5D), kasIII locus (e.g., QTL7), locus on the chromosome N l (e.g. , QTL8), and locus on the chromosome N l 9 (e.g. , QTL9), present in said first and/or second Brassica parent if present in said first or second parent,
thereby obtaining the Brassica plant.
The present disclosure further includes and provides for methods of selecting Brassica plants for the presence or absence of all or part of QTL 1 of IMC 106RR (Cargill,
Incorporated., National Registration No. 51 18); which may be used, for example, to guide breeding programs. Such methods of selecting or breeding Brassica plants comprise obtaining one or more Brassica plants and assessing their DNA to determine the presence or absence of QTL1 (on chromosome N 1 5). Based upon the results of the assessment, plants are selected for the presence or absence of all or part of QTL 1 to produce one or more selected plants.
In certain embodiments, this disclosure provides ( 1 ) canola oil having an oleic acid content of about 78% to about 80%, a linoleic acid content of about 8% to about 10%, an a- linolenic acid content of no more than about 4%, and an eicosenoic acid content of about 1.6% to about 2.3%; (2) canola oil having a total saturated fatty acid content less than about 8.5% (e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5% total saturates), and (3) canola oil having less than about 3.6% saturated fatty acids {e.g. , about 2.0% to about 3.6%, about 2.5% to about 3.6% or about 3% to about 3.6% total saturates), palmitic acid (C 16:0) content of about 6.0% or less, stearic acid (C 1 8:0) content of about 4.0% or less, arachidic acid (C20:0) content of about 1.5% or less, and/or docosanoic acid (C22:0) content of about 0.8% or less.
In other embodiments, this disclosure provides canola oil having a total saturated fatty acid content of no more than about 3.7% and an oleic acid content of about 62% to about 85% (e.g. , about 62% to about 65%, about 65% to about 72%, about 72% to about 75%, about 75% to about 80%, about 80% to about 84% and/or about 82% to about 85%). The oil may further comprise about 3.6% to about 5.2% of palmitic acid, and/or about 1.3% to about 3.0% of stearic acid. The oil may further comprise about 0.60% to about 1.10% of arachidic acid, about 0.40% to about 0.65% of docosanoic acid, and/or about 1 .6% to about 1 .9% of eicosenoic acid. The total saturated fatty acid content may be about 3.4% to about 3.7%.
In certain embodiments, the Brassica plants, including parts thereof (e.g., plant cells) and plant progeny thereof (e.g., plant seeds), disclosed herein are not genetically modified (i.e., they are non-transgenic), making them particularly useful for producing canola oils for use in food applications. In other embodiments, they may comprise only transgenes for herbicide tolerance.
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 invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be l imiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
DESCRIPTION OF DRAWINGS
FIG. 1 shows the total saturated fatty acid content in the SE 1 double haploid population (n=225). Unless specifically indicated otherwise, like reference symbols in the various drawings indicate like elements.
DETAILED DESCRIPTION
1.0 Definitions
As used herein, the term "plant" or "plants" includes parts thereof and progeny, i.e. , descendants of a particular plant or plant line, as well as cells or tissues from the plant, unless stated otherwise. Parts of plants include, but are not limited to, any one or more of: a leaf, pollen, an ovule, an embryo, a cotyledon, a hypocotyl, a meristematic cell, callus, a microspore, a root, a root tip, a pistil, an anther, a flower, a seed, a shoot, a stem, a pod, petiole and a cell or protoplast of any thereof. Progeny of an instant plant include seeds formed on Fi , F2, F3, F4 and subsequent generation plants, or seeds formed on BC |, BC2, BC3 and subsequent generation plants. Seeds produced by a plant can be grown and then selfed (or outcrossed and selfed, or doubled through DH) to obtain seeds homozygous for a mutant allele.
As used herein, the term "allele" or "alleles" refers to one or more alternative forms of a locus.
As used herein, the term "mutant alleles" or "mutation" of alleles include alleles having one or more mutations, such as insertions, deletions, stop codons, base changes (e.g. , transitions or transversions), or alterations in splice junctions, which give rise to altered gene products. Modifications in alleles may arise in coding or non-coding regions (e.g. , promoter regions, exons, introns or splice junctions).
As used herein, a "line" is a group of plants that display little or no genetic variation between individuals for at least one trait. Such lines may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques.
As used herein, the term "variety" refers to a line which is used for commercial production, and includes hybrid varieties and open-pollinated varieties.
As used herein: 24:0 or C24:0 refers to lignoceric acid; 22:0 or C22:0 refers to behenic acid; 20:0 or C20:0 refers to arachidic acid; 1 8:0 or C 1 8:0 refers to stearic acid; 16:0 or C 16:0 refers to palmitic acid; and 14:0 or C 14:0 refers to myristic acid, the terminal carboxyl groups of any of which may or may not be esterified unless indicated otherwise. As used herein, the terms "total saturated fatty acid content," "total sats" or "sats" refer to the total of myristic acid (C 14:0), palmitic acid (C 16:0), stearic acid (C 1 8:0), arachidic acid (C20:0), behenic acid (C22:0), and lignoceric acid (C24:0).
As used herein, the term "total polyunsaturates" refers to the total of linoleic acid and a-linolenic acid.
"Transgenic" or "genetically modified organisms" (GMOs) as used herein are organisms whose genetic material has been altered using techniques generally known as "recombinant DNA technology". Recombinant DNA technology is the ability to combine D A molecules from different sources into one molecule ex vivo (e.g. , in a test tube). This terminology generally does not cover organisms whose genetic composition has been altered by conventional cross-breeding or by "mutagenesis" breeding, as these methods predate the discovery of recombinant DNA techniques. See, World Health Organization, Biorisk management Laboratory biosecurity guidance, 2006 World Health Organization
(WHO/CDS/EPR/2006.6).
"Non-transgenic" as used herein refers to plants and food products derived from plants that are not "transgenic" or "genetically modified organisms" as defined above. The plants described herein are non-transgenic to the extent that they are derived by mutagenesis.
As used herein, the term "position" refers to the location of the SNP by base pair within the pseudo molecules of Brassica oleracea genome (B. oleracea TO 1000 version 4; released date is 12-Jan-2012) obtained from Canseq consortium (http://aafc- aac.usask.ca/canseq/).
As used herein, the term "weight percent," "percent by weight," or "wt%" of a fatty acid refers to the percent by weight of the fatty acids having from 14 carbon atoms (C 14 fatty acids) to 24 carbon atoms (C24 fatty acids). When used in connection with a seed, the term refers to the percent by weight of the total of those fatty acids in the seed oil fraction.
As used herein, the term "sequence identity" refers to the degree of identity between any given nucleic acid sequence and a target nucleic acid sequence. Percent sequence identity is calculated by determining the number of matched positions in aligned nucleic acid sequences, dividing the number of matched positions by the total number of aligned nucleotides, and multiplying by 100. A matched position refers to a position in which identical nucleotides occur at the same position in aligned nucleic acid sequences. Percent sequence identity also can be determined for any amino acid sequence. To determine percent sequence identity, a target nucleic acid or amino acid sequence is compared to the identified nucleic acid or amino acid sequence using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN and BLASTP. This stand-alone version of BLASTZ can be obtained from Fish & Richardson's web site (World Wide Web at fr.com/blast) or the U.S. government's National Center for Biotechnology Information web site (World Wide Web at ncbi.nlm.nih.gov). Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ. BI2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g. , C:\seq l .txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g. , C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g. , C :\output.txt); -q is set to - 1 ; -r is set to 2; and all other options are left at their default setting. The following command will generate an output file containing a comparison between two sequences: C:\B12seq -i c:\seql .txt -j c:\seq2.txt -p blastn -o c:\output.txt -q - 1 -r 2. If the target sequence shares homology with any portion of the identified sequence, then the designated output file will present those regions of homology as aligned sequences. If the target sequence does not share homology with any portion of the identified sequence, then the designated output file will not present aligned sequences. Once aligned, a length is determined by counting the number of consecutive nucleotides from the target sequence presented in alignment with the sequence from the identified sequence starting with any matched position and ending with any other matched position. A matched position is any position where an identical nucleotide is presented in both the target and identified sequences. Gaps presented in the target sequence are not counted since gaps are not nucleotides. Likewise, gaps presented in the identified sequence are not counted since target sequence nucleotides are counted, not nucleotides from the identified sequence. The percent identity over a particular length is determined by counting the number of matched positions over that length and dividing that number by the length followed by multiplying the resulting value by 100. For example, if (i) a 500-base nucleic acid target sequence is compared to a subject nucleic acid sequence, (ii) the B12seq program presents 200 bases from the target sequence aligned with a region of the subject sequence where the first and last bases of that 200-base region are matches, and (iii) the number of matches over those 200 aligned bases is 180, then the 500-base nucleic acid target sequence contains a length of 200 and a sequence identity over that length of 90% (i.e. , 180 / 200 x 100 = 90). It will be appreciated that different regions within a single nucleic acid target sequence that al igns with an identified sequence can each have their own percent identity. It is noted that the percent identity value is rounded to the nearest tenth. For example, 78.1 1 , 78.12, 78.13, and 78.14 are rounded down to 78. 1 , while 78.15, 78.16, 78.1 7, 78.18, and 78.19 are rounded up to 78.2. It also is noted that the length value will always be an integer.
2.0 Development of Fatty Acid Traits and Their Molecular Mapping
Cross breeding of plants and subsequent mapping of the DNA markers associated with the genetic traits permits the identification of the genetic basis for the traits. Mapping of genomic blocks responsible for traits also assists in the effective transfer of the traits into other members of the genus and/or species, including elite production lines with other desirable characteristics (e.g. , disease resistance, herbicide tolerance, drought resistance...).
2.1 QTL Mapping
Mapping of QTL1 has been accomplished using the DH population of 225 lines derived from a cross between IMC 106RR and the biennial variety Wichita. Mapping in the population permits localization of QTL 1 on chromosome N 15 that explains a significant proportion of the variation in C 18:0, C20:0, C22:0 and total saturates (Table 1 ). DH lines carrying the IMC 106RR allele are significantly lower in each fatty acid component than DH lines carrying the Wichita al lele. Analysis of the QTL 1 region using the single marker regression approach identifies the marker located at 42605157 bp on C05 of the C genome as the most highly correlated (the highest percent variance explained (R2), Table 2, based on a C genome assembly). Further examination of mean trait values of lines carrying alternative alleles at 42877318 bp shows a further reduction in mean values of all traits in lines carrying the IMC 106RR allele (Table 3), suggesting this locus may be closer to the causal genetic variant than the original. This location is considered the "center" of the QTL 1 which encompass a 2 Mb region having 1 Mb on either side of the center. The "center" QTL 1 marker (at 42877318 bp on C05 of the C genome) is located at 44730765 bp on N l 5 of the DH 12075 Brassica napus genome assembly (see CanSeq Consortium website at http://aafc- aac.usask.ca canseq ). Accordingly, in terms of the B. napus genome, and for the purposes of physical locations for the NGS marker list, the QTL 1 interval is defined as spanning from 43730765^15730765 bp on N 15 of the DH 12075 B. napus genome assembly. 2.2 Sequencing, Alignment, and Polymorphism Analysis
Mapping of the genomic sequencing data (e.g., fastq files) to a Brassica napus reference genome (19 linkage groups of B. napus genotype DH 12075, CanSeq Consortium) is performed using SeqMan NGen v4 (DNAStar, Madison, WI). The alignment is performed using default settings for read mapping and SNP calling. A list of 38 SNPs is generated (Table 4), requiring all selected SNPs to be homozygous variant and to be unique to the IMC 106R.R genotype (i .e., different than the Wichita sequence and the DH 12075 reference sequence).
Examination of the 1 5 QTL I interval identified several candidate genes that may be responsible for the observed phenotype (Table 5). These genes are located within the QTL I interval, and selected as candidate genes based upon their integral activity in plant lipid biosynthetic pathways.
3.0 QTLI
In certain embodiments, the Brassica plants provided herein are non-transgenic. In other embodiments, they may comprise only transgenes for herbicide tolerance. Examples of the Brassica plants include, without limitation, B. napus, B. juncea, B. rapa, B. oleracea, and B. carinata. The Brassica plants yield seed oil with a total saturated fatty acid content of about 7% or less, or having a total saturated fatty acid content of about 3.6% or less, that comprise one or more mutations at a locus termed QTL I on chromosome N l 5. This locus is defined by its correlation to the variation of C 18:0 and/or C20:0 and/or C22:0 fatty acid content of seed oil and the SNP markers identified herein. QTL I is believed to reside upon B. napus chromosome N l 5 based upon the mapping populations described herein. However, it is understood that the locus is defined not by chromosomal position but by its SNP markers and its correlation to the fatty acids content of seed oil, and therefore the locus may appear on other chromosomes, particularly in progeny. The appearance of QTL I on other
chromosomes may result from a variety of events including, but not limited to, homologous chromosomal crossover events. The occurrence of crossover events may be higher in plants such as B. napus, which is an allopolyploid species.
Accordingly, in various embodiments, provided herein is a Brassica plant or a part thereof comprising a nucleic acid sequence having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences between the chromosome N 1 5 (QTL 1 ) SNP markers at positions 43730765 and 45730765 of the B. napus line 1 C 106RR, wherein said part of the genomic sequences of the B. napus 1MC 106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides. In certain embodiments, the Brassica plant or a part thereof is transgenic; in certain embodiments, the Brassica plant or a part thereof is non-transgenic; and in other embodiments, the Brassica plant or a part thereof comprises only transgenes for herbicide tolerance.
In another embodiment, the Brassica plant, or a part thereof, comprises a nucleic acid sequence having greater than about 80% (e.g., about 85%), about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%), about 99.995%, or about 99.999%) identity to all or part of the genomic sequence of the B. napus line IMC 106RR between positions 43730765 and 45730765 and/or any two SNP markers between those positions, wherein the SNP markers between those positions are selected from: 43849348, 43877053, 44223 135, 44252880, 44407747, 44617229, 44659768, 44736857, 44746871 , 44756604, 44771673, 44794771 , 44796526, 44798855, 44839954, 44855978, 44859545, 44872594, 44923355, 44985743, 45007425, 45093354, 45147723, 45159244, 45165499, 45256038, 45260347, 45278355, 45298286, 45312516, 45354975, 45365977, 45378346, 45402371 , 45409080, 45496931 , 45499138, and 4572071 5, wherein said part of the genomic sequences of the B. napus I C 106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
In certain embodiments, the Brassica plants comprise one, two, three, four, five, ten, fifteen or more QTL 1 markers selected from the group consisting of:
43849348, 43877053, 442231 35, 44252880, 44407747, 44617229, 44659768, 44736857, 44746871 , 44756604, 44771673, 44794771 , 44796526, 44798855, 44839954, 44855978, 44859545, 44872594, 44923355, 44985743, 45007425, 45093354, 45147723, 451 59244, 45165499, 45256038, 45260347, 45278355, 45298286, 453 12516, 45354975, 45365977, 45378346, 45402371 , 45409080, 4549693 1 , 45499138, and 4572071 5.
In certain embodiments, the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%)) identity to all or part of the genomic sequences within the segments between any two chromosome N l 5 (QTL 1 ) SNP markers at positions selected from: 43849348, 43877053, 44223135, 44252880, 44407747, 44617229, 44659768, 44736857, 44746871 , 44756604, 44771673, 44794771 , 44796526, 44798855, 44839954, 44855978, 44859545, 44872594, 44923355, 44985743, 45007425, 45093354, 45147723, 45159244, 45165499, 45256038, 45260347, 45278355, 45298286, 45312516, 45354975, 45365977, 45378346, 45402371 , 45409080, 4549693 1 , 45499138, and 4572071 5 of the genomic sequence of B. napus line IMC 106RR (Cargill, Incorporated, National Registration No. 51 1 8), wherein said part of the genomic sequences of the B. napus IMC 106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
In certain embodiments, the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences comprising any one or more (e.g., two, three, or four) chromosome N l 5 (QTL 1 ) SNP markers at positions selected from: 43849348, 43877053, 44223135, 44252880, 44407747, 44617229, 44659768, 44736857, 44746871 , 44756604, 44771673, 44794771 , 44796526, 44798855, 44839954, 44855978, 44859545, 44872594, 44923355, 44985743, 45007425, 45093354, 45147723, 451 59244, 45 165499, 45256038, 45260347, 45278355, 45298286, 4531 2516, 45354975, 45365977, 45378346, 45402371 , 45409080, 45496931 , 45499138, and 45720715 of the genomic sequence of B. napus line IMC 106RR (Cargill, Incorporated, National Registration No. 51 18), wherein said part of the genomic sequences of the B. napus IMC 106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides. In certain embodiments, the nucleic acid comprises one or more sequences selected from the group consisting of SEQ ID NOs: 2- 1 , 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2- 10, 2- 1 1 , 2- 12, 2- 13, 2- 14, 2- 15, 2- 16, 2- 1 7, 2- 1 8, 2- 19, 2-20, 2-21 , 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31 , 2-32, 2-33, 2-34, 2- 35, 2-36, 2-37, and 2-38.
In certain embodiments, the Brassica plants comprise a nucleic acid sequence having greater than 80% identity to all or part of the genomic sequences between the chromosome N l 5 (QTL 1 ) SNP markers at positions 43849348 and 4572071 5 of the genomic sequence of B. napus IMC 106RR line, wherein said part of the genomic sequences of the B. napus IMC 106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
In certain embodiments, the Brassica plants comprise one, two, or three single nucleotide polymorphisms on B. napus chromosome N 1 5 selected from the group consisting of C to T transitions identified in the B. napus I MC 106RR line, National Registration No. 51 18, at locations: 43877053, 44659768, and 44746871 . In certain embodiments, the Brassica plants comprise one, two, or three single nucleotide polymorphisms on B. napus chromosome N l 5 selected from the group consisting of T to C transitions identified in the B. napus IMC 106RR line, National Registration No. 51 18, at locations: 45093354, 45354975, and 45378346.
In certain embodiments, the Brassica plants comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve single nucleotide polymorphisms on B. napus chromosome N l 5 selected from the group consisting of G to A transitions identified in the B. napus IMC 106RR line, National Registration No. 51 1 8, at locations: 44252880, 44407747, 44617229, 44756604, 44794771 , 44855978, 44859545, 44872594, 45007425, 45 159244, 453125 16, and 45365977.
In certain embodiments, the Brassica plants comprise one, two, three, four, five, or six single nucleotide polymorphisms on B. napus chromosome N l 5 selected from the group consisting of A to G transitions identified in the B. napus IMC 106RR line, National Registration No. 51 1 8, at locations: 44617229, 44796526, 44923355, 45147723, 45260347, and 45278355.
In certain embodiments, the Brassica plant comprises a B. napus N 15 chromosome having greater than 95%, 97.5%, 98%, 99%, 99.9%, 99.99%, 99.999% identity or having 100% identity to all or part of the genomic sequences of the B. napus I MC 106RR line, National Registration No. 51 18, on an N 15 chromosomal segment selected from the group consisting of segments:
beginning with SNP 43849348 and ending with SNP 4572071 5 or SNP 45402371 ; beginning with SNP 43849348 and ending with SNP 45312516 or SNP 45256038; beginning with SNP 43849348 and ending with SNP 45165499 or SNP 45007425; beginning with SNP 43849348 and ending with SNP 44923355 or SNP 44839954; beginning with SNP 43849348 and ending with SNP 44771673 or SNP 44659768; beginning with SNP 43849348 and ending with SNP 44407747 or SNP 44223 1 35; beginning with SNP 44223 1 35 and ending with SNP 4572071 5 or SNP 45402371 ; beginning with SNP 44223 135 and ending with SNP 45312516 or SNP 45256038; beginning with SNP 44223135 and ending with SNP 45165499 or SNP 45007425; beginning with SNP 44223135 and ending with SNP 44923355 or SNP 44839954; beginning with SNP 44223135 and ending with SNP 44771673 or SNP 44659768; and
beginning with SNP 44223 135 and ending with SNP 44407747 or SNP 44223 1 35; wherein said part of the genomic sequences of the B. napus IMC 106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
A number of candidate genes that may correlate to the variance of fatty acid profile (e.g., C 18:0 and/or C20:0 and/or C22:0 and/or total saturates) of plants are present in or tightly linked to the region into which QTL 1 has been mapped. The genes encoding B. napus (1 ) fatty acyl-CoA reductase (AT3G 1 1980); (2) glycerol-3-phosphate acyltransferase (AT3G 1 1430); (3) glycerol-3-phosphate acyltransferase (AT3G 1 1430); (4)
digalactosyldiacylglycerol synthase (AT3G 1 1670)); (5) 4'-phosphopantetheinyl transferase superfamily (AT3G 1 1470); (6) glycerol-3-phosphate acyltransferase (AT3G 1 1430); (7) fatty acid desaturase 7 (AT3G1 1 170 ); (8) alpha/beta-Hydrolases superfamily protein
(AT3G 10840); (9) Fatty Acyl in-chain Hydroxylase (AT3G 10570); ( 10)
phosphatidylinositol-4-phosphate 5-kinase family protein (AT3G 14270); ( 1 1 ) FAD- dependent oxidoreductase family protein (AT3G 10370); ( 12) phosphatidyl inositol monophosphate 5 kinase (AT3G09920); ( 1 3) phosphatidic acid phosphohydrolase 1 (AT3G09560); ( 14) lipid transfer protein 6 (AT3G08770); ( 1 5) phospholipase C 2
(AT3G08510); ( 16) 6-phosphogluconate dehydrogenase family protein (AT3G07690); ( 17) bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin superfamily protein (AT3G07450) and ( 18) lipase class 3 family protein (AT3G07400) are among the genes present in the interval, which, as previously indicated, is believed to be on chromosome N 1 5, onto which QTL 1 has been mapped.
Brassica plants described herein may produce a seed oil having a total saturated fatty acid content of about 2.5% to about 5.5%, about 3% to about 5%, about 3% to about 4.5%, about 3.25% to about 3.75%, about 3% to about 3.5%, about 3.6% to about 5%, about 4% to about 5.5%, or about 4% to about 5%. Oi ls having total saturated fatty acid content of about 8.5% or lower are perceived to have improved nutritional quality and can help consumers reduce their intake of saturated fatty acids.
Brassica plants having one or more mutations at QTL 1 locus described herein can yield a seed oil having a total saturated fatty acid content of about 8.5% or lower (e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5% total saturates), in combination with about 60% to about 70%, about 71 % to about 80%, or more than about 80% oleic acid content. Such Brassica plants can produce a seed oil having a fatty acid content tailored to the desired end use of the oil (e.g. , frying or other food applications). For example, Brassica plants can be produced that yield a seed oil having a total saturated fatty acid content of about 8.5% or lower, an oleic acid content of about 60% to about 70%, and an a-linolenic acid content of about 2% to about 5%. Total polyunsaturates in such seed oi ls typically are less than about 30%, preferably less than about 28%, e.g. , about 1 5% to about 27.5%, about 17% to about 27%, or about 20% to about 25%. Canola oils having such fatty acid contents are particularly useful for frying applications due to the polyunsaturated content, which is low enough to provide improved oxidative stability for frying, yet high enough to impart the desired fried flavor to the food being fried, and are an improvement over commodity type canola oils. By comparison, the fatty acid content of commodity type canola oils typically is about 6% to about 8% total saturated fatty acids, about 55% to about 65% oleic acid, about 22% to about 30% linoleic acid, and about 7% to about 10% a- linolenic acid.
Brassica plants described herein also may yield a seed oil having a total saturated fatty acid content of about 1.6% to about 3%, about 2% to about 4%, about 3% to about 6%, about 71% to about 80%, or about 2% to about 5.0%. Canola oils having such fatty acid contents have higher oxidative stabilities than oils with a lower oleic acid and higher α-linolenic acid content and/or commodity type canola oils. Thus, such canola oils are useful for coating applications (e.g. , spray-coatings), formulating food products, or other applications where shelf-life stability is desired. In addition, Brassica plants described herein can yield a seed oi l having a total saturated fatty acid content of less than about 8.5% {e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5% total saturates), an oleic acid content of about 81 % to about 90% and an α-linolenic acid content of about 2% to about 5%. Canola oils having a total saturated fatty acid content less than about 8.5% {e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5% total saturates), an oleic acid content of about 81 % to about 90%, and an α-linolenic acid content of about 2% to about 5% are particularly useful for food applications requiring high oxidative stability and a reduced saturated fatty acid content.
4.0 Molecular Guided Breeding
The present disclosure further includes and provides for methods of selecting or breeding Brassica plants for the presence or absence of all or part of QTLI of IMC 106RR (Cargill, Incorporated, National Registration No. 51 1 8) that may be employed, for example, as molecular guided breeding programs. Such methods of selecting or breeding Brassica plants comprise obtaining one or more Brassica plants and assessing their DNA to determine the presence or absence of all or part of QTLI (on chromosome NI5). Based upon the results of the assessment, plants are selected for the presence or absence of all or part of QTLI to produce one or more selected plants. Such methods may be used, for example, to determine which progeny resulting from a cross have all or part of QTLI, and accordingly to guide preparation of plants having one or both of those QTLs in combination with other desirable genes/traits.
In one embodiment, determining the presence of all or part of QTL I in plants comprises determining the presence of mutations appearing in IMC 106 R in the QTL 1 region that do not appear in its parent. Accordingly, plants can be selected by assessing them for the presence of one or more individual SNPs appearing in Table 4 for QTL I . Plants may also be assessed for larger portions of those QTL regions (e.g., regions encompassing one or more SNPs in Table 4).
In certain embodiments, plants may be selected by determining the presence of one, two, three, four, five, ten, fifteen or more QTL I markers selected from the group consisting of: 43849348, 43877053, 44223 135, 44252880, 44407747, 4461 7229, 44659768, 44736857, 44746871 , 44756604, 44771673, 44794771 , 44796526, 44798855, 44839954, 44855978, 44859545, 44872594, 44923355, 44985743, 45007425, 45093354, 45147723, 45159244, 45165499, 45256038, 45260347, 45278355, 45298286, 45312516, 45354975, 45365977, 45378346, 45402371 , 45409080, 45496931 , 45499138, and 45720715.
In certain embodiments, plants may be assessed to determine the presence or absence of QTLI chromosomal segments including a segment selected from the chromosomal regions:
beginning w th SNP 43849348 and ending with SNP 4572071 5 or SNP 4540237 1 beginning w th SNP 43849348 and ending with SNP 453 125 16 or SNP 45256038 beginning w th SNP 43849348 and ending with SNP 45 165499 or SNP 45007425 beginning w th SNP 43849348 and ending with SNP 44923355 or SNP 44839954 beginning w th SNP 43849348 and ending with SNP 44771673 or SNP 44659768 beginning w th SNP 43849348 and ending with SNP 44407747 or SNP 44223135 beginning w th SNP 44223 135 and ending with SNP 45720715 or SNP 45402371 beginning w th SNP 44223135 and ending with SNP 45312516 or SNP 45256038 beginning w th SNP 44223135 and ending with SNP 45165499 or SNP 45007425 beginning w th SNP 44223135 and ending with SNP 44923355 or SNP 44839954 beginning w th SNP 44223 135 and ending with SNP 44771673 or SNP 44659768 and beginning with SNP 44223135 and ending with SNP 44407747 or SNP 442231 35.
In certain embodiments, plants may be assessed to determine the presence or absence of QTL 1 SNP.
Any suitable method known in the art may be used to assess plants to determine if they comprise all or part of QTL1 . Some suitable methods include, but are not limited to, sequencing, hybridization assays, polymerase chain reaction (PCR), ligase chain reaction (LCR), and genotyping-by-sequencing (GBS).
In addition to selecting plants based upon the presence or absence of all or part of QTL1 , the plants may be assessed for their fatty acid content. More specifically, plants may be assessed for their fatty acid profile (i.e., the types and/or relative amount of fatty acids they produce, typically in their seed) and their total fatty acid production. Among the fatty acids that can be examined are saturated fats (e.g., 16:0, 18:0, 20:0, etc.), monounsaturated fats, and polyunsaturated fats. Analysis of fatty acid profile and/or content may be directed to one or more selected plants (or their seed) selected and/or the progeny of such plants. In some embodiments, the Brassica plants described herein comprise one or more alleles QTL 1 of the IMC 106RR line and further comprise a mutant allele for a fatty acyl-ACP thioesterase. Fatty acyl-ACP thioesterases hydrolyze acyl-ACPs in the chloroplast to release the newly synthesized fatty acid from ACP, effectively removing it from further chain elongation in the plastid. The free fatty acid can then leave the plastid, become bound to CoenzymeA (CoA) and enter the Kennedy pathway in the endoplasmic reticulum (ER) for triacylglycerol (TAG) biosynthesis. Members of the FATA family prefer oleoyl (C 18: 1 ) ACP substrates with minor activity towards 18:0 and 16:0 ACPs, while members of the FATB family hydrolyze primarily saturated acyl-ACPs between 8 and 18 carbons in length. See Jones et al., Plant Cell 7:359- 371 , 15 (1 995); Ginalski and Rychlewski, Nucleic Acids Res 31 :3291 -3292 (2003); and Voelker T in Genetic Engineering (Setlow, JK, ed) Vol 1 8, 1 1 1 - 1 33, Plenum Publishing Corp., New York (2003).
In certain embodiments, provided herein a method of DNA-assisted selection of a reduced-palmitic acid oi l trait in Brassica plants, comprising:
a) assessing the DNA of the Brassica plants to determine the presence or absence of all of, or a part comprising greater than 20, 30, 40, 50 or 60 contiguous nucleotides of,
QTL 1 ; and
b) selecting one of the Brassica plants in which QTL 1 , or at least said part of QTL 1 , is present. In certain embodiments, the method of DNA-assisted selected described supra further comprises selecting a second one of the Brassica plants in which QTL 1 , or at least said part of QTL1 , is present.
In certain embodiments, the method of DNA-assisted selected described supra further comprises selecting a second one of the Brassica plants in which QTL 1 , or at least said part of QTL1 , is present, and:
a) crossing each of the selected Brassica plants with a second canola plant to produce a plurality of F l Brassica plants
b) assessing the DNA of the F l Brassica plants to determine the presence or absence of QTL 1 ; and
c) selecting one of the F l Brassica plants in which QTL 1 , or at least said part of QTL 1 , is present.
In certain embodiments, the method of DNA-assisted selected described supra further comprises:
a) crossing the selected canola plant with a second canola plant to produce a F l
Brassica plant; and
b) assessing the DNA of the F l Brassica plant to determine the presence or absence of QTL 1.
5.0 Brassica plants comprising reduced activities of certain enzymes related to the production of saturated and unsaturated fatty acids.
5.1 Fatty acyl-ACP thioesterases
Fatty acyl-ACP thioesterases hydrolyze acyl-ACPs in the chloroplast to release the newly synthesized fatty acid from ACP, effectively removing it from further chain elongation in the plastid. The free fatty acid can then leave the plastid, become bound to CoenzymeA (CoA) and enter the Kennedy pathway in the endoplasmic reticulum (ER) for triacylglycerol (TAG) biosynthesis. Members of the FATA family (e.g. , FATA2) prefer oleoyl (C 18: 1 ) ACP substrates with minor activity towards 18:0 and 16:0 ACPs, while members of the FATB family (e.g., FATB) hydrolyze primarily saturated acyl-ACPs between 8 and 1 8 carbons in length. See Jones et al., Plant Cell 7:359-371 ( 1 995); Ginalski and Rychlewski, Nucleic Acids Res 31 :3291 -3292 (2003); and Voelker T in Genetic Engineering (Setlow, JK, ed) Vol 1 8, 1 1 1 - 1 33, Plenum Publishing Corp., New York (2003).
Reduced activities, including absence of detectable activity, of FATA2, and/or FATB may be achieved by modifying an endogenous fatA2 and/or fatB allele(s), respectively. An endogenous fatA2 or fat3B allele can be modified by, for example, mutagenesis or by using homologous recombination to replace an endogenous plant gene with a variant containing one or more mutations (e.g. , produced using site-directed mutagenesis). See, e.g. , Townsend et al., Nature 459:442-445 (2009); Tovkach et al., Plant J, 57:747-757 (2009); and Lloyd et al., Proc. Natl. Acad. Sci. USA, 102:2232-2237 (2005). Similarly, for other genes discussed herein, the endogenous allele can be modified by mutagenesis or by using homologous recombination to replace an endogenous gene with a variant. Alleles comprising mutations obtained through mutagenesis are referred to as mutant alleles herein.
Reduced activities, including absence of detectable activity, can be inferred from the decreased level of saturated fatty acids in the seed oil compared with the seed oil from a control plant. In one embodiment, the Brassica l ine Topas, ATCC deposit 40624, may serve as a control plant. To ensure the continued availability of B. napus cv. Topas as a reference, it has been redeposited and designated ATCC deposit PTA- 120738. Alternatively, reduced activities can be assessed in plant extracts using assays for fatty acyl-ACP hydrolysis. See, for example, Bonaventure et al., Plant Cell 15: 1020- 1033 (2003); and Eccleston and Ohlrogge, Plant Cell 10:613-622 (1998).
Genetic mutations can be introduced within a population of seeds or regenerable plant tissue using one or more mutagenic agents. Suitable mutagenic agents include, for example, ethyl methane sulfonate (EMS), methyl N-nitrosoguanidine (MN G), ethidium bromide, diepoxybutane, ionizing radiation, x-rays, UV rays and other mutagens known in the art. In some embodiments, a combination of mutagens, such as EMS and MNNG, can be used to induce mutagenesis. The treated population, or a subsequent generation of that population, can be screened for specific enzyme activities (e.g., reduced thioesterase or acyltransferase activity) that result from the mutation(s), e.g. , by determining the fatty acid profile of the population and comparing it to a corresponding non-mutagenized population. Mutations can be in any portion of a gene, including coding sequence, intron sequence and regulatory elements, that renders the resulting gene product non-functional or with reduced activities. Such mutations can lead to deletion or insertion of amino acids, and conservative or non- conservative amino acid substitutions in the corresponding gene product. In some embodiments, the mutation is a nonsense mutation, which results in the introduction of a stop codon (TGA, TAA, or TAG) and production of a truncated polypeptide. In some embodiments, the mutation is a splice site mutation which alters or abolishes the correct splicing of the pre-mRNA sequence, resulting in a protein of different amino acid sequence than the wild type. For example, one or more exons may be skipped during RNA splicing, resulting in a protein lacking the amino acids encoded by the skipped exons. Alternatively, the reading frame may be altered by incorrect splicing, one or more introns may be retained, alternate splice donors or acceptors may be generated, splicing may be initiated at an alternate position, or alternative polyadenylation signals may be generated. In some embodiments, more than one mutation or more than one type of mutation is introduced.
Insertions, deletions, or substitutions of amino acids in a coding sequence may, for example, disrupt the conformation of essential alpha-helical or beta-pleated sheet regions of the resulting gene product. Amino acid insertions, deletions, or substitutions also can disrupt binding, alter substrate specificity, or disrupt catalytic sites important for gene product activity. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product non-functional, compared to a smaller number of inserted or deleted amino acids. Non-conservative amino acid substitutions may replace an amino acid of one class with an amino acid of a different class. Non-conservative substitutions may make a substantial change in the charge or hydrophobicity of the gene product. Non-conservative amino acid substitutions may also make a substantial change in the bulk of the residue side chain, e.g. , substituting an alanine residue for an isoleucine residue.
Examples of non-conservative substitutions include the substitution of a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid. Because there are only 20 amino acids encoded in a gene, substitutions that result in reduced activity may be determined by routine experimentation, incorporating amino acids of a different class in the region of the gene product targeted for mutation.
In addition to mutations in protein coding sequences, mutations may arise in the non- coding portions of genes, such as the promoter regions that alter the plants ability to express normal amounts of the mRNA and the protein it encodes. Mutations may also arise that increase the activity of proteins such as, for example, mutations that favor the pathways consistent with the saturated fatty acid profiles described herein for Brassica plants bearing QTL1.
Mutations in fatA2 and fatB alleles in Brassica plants have previously been described as useful in controlling the total saturated fatty acid content in the seed oil of plants of Brassicaceae, see e.g. , WO 201 1 /075716, herein is incorporated by reference in its entirety. 5.1.1 Fatty acyl-ACP thioesterase A2 (FATA2)
In some embodiments, the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus I MC 106RR and further comprise one or more mutant alleles at one fatA2 locus, wherein the one or more mutant alleles result in the production of a FATA2 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2 polypeptide (SEQ ID NO: QTL3A.p, wherein the position corresponding to position 255 as numbered based on the alignment to the Arabidopsis thaliana FATA2 polypeptide set forth in GenBank Accession No. NP_1 93041 .1 , protein (SEQ ID NO:2.p); GenBank Accession No. NM_1 1 7374, mRNA is leucine).
Where a mutant allele at a fatA2 locus (e.g. QTL3A) is present, the mutant allele may comprise a nucleic acid that encodes a FATA2 polypeptide having a non-conservative substitution within a helix/4-stranded sheet (4HBT) domain (also referred to as a hot-dog domain) or a non-conservative substitution of a residue affecting catalytic activity or substrate specificity. In one example, the mutant allele at the fatA2 locus (QTL3A) comprises a nucleic acid encoding a FATA2 polypeptide having a substitution in a region (SEQ ID: QTL3A.p, wherein the position corresponding to position 255 as numbered based on the alignment to the Arabidopsis thaliana FATA2 polypeptide set forth in GenBank Accession No. NP_193041 .1 , protein (SEQ ID NO:2.p); GenBank Accession No.
NM_1 17374, mRNA is leucine) of the polypeptide corresponding to residues 242 to 277 of the FATA2 polypeptide (as numbered based on the alignment to the Arabidopsis thaliana FATA2 polypeptide set forth in GenBank Accession No. NP_193041 . 1 , protein (SEQ ID NO:2.p); GenBank Accession No. NM_1 17374, mRNA). Examples of such a nucleic acid include, without limitation, the nucleic acid sequences of SEQ ID NO: QTL3A.n, wherein nucleotide 1387 is T. This region of FATA2 is highly conserved in Arabidopsis and Brassica. In addition, many residues in this region are conserved between FATA and FATB, including the aspartic acid at position 259, asparagine at position 263, histidine at position 265, valine at position 266, asparagine at position 268, and tyrosine at position 271 (as numbered based on the alignment to SEQ ID NO:2.p). The asparagine at position 263 and histidine at position 265 are part of the catalytic triad, and the arginine at position 256 is involved in determining substrate specificity. See also Mayer and Shanklin, BMC Plant
Biology 7: 1 -1 1 (2007). SEQ ID: QTL3A.p sets forth the predicted amino acid sequence of the Brassica FATA2 polypeptide. For example, the FATA2 polypeptide can have a substitution of a leucine residue for proline at the position corresponding to position 255 of the Arabidopsis FATA2 polypeptide (i.e. , SEQ ID NO: 2.p and QTL3A.p). The proline in the B. napus sequence corresponding to position 255 in Arabidopsis is conserved among B. napus, B. rapa, B. juncea, Zea mays, Sorghum bicolor, Oryza sativa Indica (rice), Triticum aestivum, Glycine max, Jatropha (tree species), Carthamus tinctorius, Cuphea hookeriana, Iris tectorum, Perilla frutescens, Helianthus annuus, Garcinia mangostana, Picea sitchensis, Physcomitrella patens subsp. Patens, Elaeis guineensis, Vitis vinifera, Elaeis oleifera, Camellia oleifera, Arachis hypogaea, Capsicum annuum, Populus trichocarpa, and
Diploknema butyracea. The mutation at position 255 is associated with a phenotype producing an oil with a total saturated fatty acid content of about 8.5% or lower (e.g. , about 3.6% to about 8.5%, about 5.0% to about 8.5%, or about 7% to about 8.5% total saturates), stearic acid content of about 4.0% or lower, arachidic acid content of about 1 .5% or lower, and an increased eicosenoic acid phenotype. The stearic acid content phenotype is negatively correlated with the eicosenoic acid phenotype.
In some embodiments, where one or more mutant alleles at one fatA2 locus (e.g. QTL3A) are present, the loci have at least about 90% (e.g. , at least about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9% or about 99.99%) sequence identity to the nucleotide sequence set forth in QTL3A The nucleotide sequences set forth in QTL3A are representative nucleotide sequences from the fatA2 gene from B. napus line 15.24.
5.1.2 Fatty acyl-ACP thioesterase B (FATB).
Brassica napus contains 6 different fatB isoforms (i.e. , different forms of the FATB polypeptide at different loci), which are designated isoforms 1 -6 herein. In some embodiments, the Brassica plants described herein are homozygous or heterozygous for B. napus I MC 106RR QTL l and further comprise one or more mutant alleles at one or more fatB loci, wherein the mutant allele(s) at the one or more fatB loci results in the production of a
FATB polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATB polypeptide (SEQ ID : QTL5A.p, QTL5B.p, QTL5C.p and QTL5D.p). In some embodiments, the Brassica plant contains one or more mutant alleles at two or more different fatB loci. In some embodiments, the Brassica plant contains one or more mutant alleles at three different fatB loci. In some embodiments, the Brassica plant contains mutant alleles at four different fatB loci.
For example, the Brassica plant comprising a fatB mutation can have a nucleotide sequence encoding FATB isoform 1 , isoform 2, isoform 3, or isoform 4. In some embodiments, the plant can have a nucleotide sequence encoding isoforms 1 and 2; 1 and 3; 1 and 4; 2 and 3; 2 and 4; 3 and 4; 1 , 2, and 3; 1 , 2, and 4; 2, 3, and 4; or 1 , 2, 3, and 4;. I n some embodiments, the Brassica plant can have a mutation in nucleotide sequences encoding FATB isoforms 1 , 2, and 3; 1 , 2, and 4; 2, 3, and 4; or 1 , 2, 3, and 4. In some embodiments, a mutation results in deletion of a 4HBT domain or a portion thereof of a FATB polypeptide. In addition, the plants may be homozygous or heterozygous for the mutant alleles of each isoform.
FATB polypeptides typically contain a tandem repeat of the 4HBT domain, where the N-terminal 4HBT domain contains residues affecting substrate specificity (e.g. , two conserved methionines, a conserved lysine, a conserved valine, and a conserved serine) and the C-terminal 4HBT domain contains residues affecting catalytic activity (e.g. , a catalytic triad of a conserved asparagine, a conserved histidine, and a conserved cysteine) and substrate specificity (e.g. , a conserved tryptophan). See Mayer and Shanklin, J. Biol. Chem. 280:3621 -3627 (2005). In some embodiments, the mutation results in a non-conservative substitution of a residue in a 4HBT domain or a residue affecting substrate specificity. In some embodiments, the mutation is a splice site mutation. In some embodiments, the mutation is a nonsense mutation in which a premature stop codon (TGA, TAA, or TAG) is introduced, resulting in the production of a truncated polypeptide.
SEQ ID NOs: QTL5A.n, QTL5B.n, QTL5C.n and QTL5D.n set forth the nucleotide sequences encoding FATB isoforms 1 -4, respectively, and containing exemplary nonsense mutations that result in truncated FATB polypeptides. SEQ ID NO: QTL5A.n is the nucleotide sequence of isoform 1 having a mutation at position 154, which changes the codon from CAG to TAG. SEQ ID NO: QTL5B.n is the nucleotide sequence of isoform 2 having a mutation at position 695, which changes the codon from CAG to TAG. SEQ ID NO:
QTL5C.n is the nucleotide sequence of isoform 3 having a mutation at position 276, which changes the codon from TGG to TGA. SEQ I D NO: QTL5D.n is the nucleotide sequence of isoform 4 having a mutation at position 336, which changes the codon from TGG to TGA.
5.2 Fatty acid desaturases that modulate oleic acid and linolenic acid levels 5.2.1 Delta-12 desaturase (FAD2)
In other embodiments, Brassica plants homozygous or heterozygous for QTL l of
IMC 106RR may also have decreased activity of FAD2, which is involved in the enzymatic conversion of oleic acid to linoleic acid, to confer an oleic acid content of about 70% to about 80% or about 80% or higher in the seed oil. The sequences for the wild-type fad2 genes from B. napus (termed the D form and the F form) are disclosed in W098/56239, which is incorporated herein by reference (SEQ ID NO: QTL4A.n, wherein nucleotide 3 16 is G, and SEQ ID NO: QTL4B.n, wherein nucleotide 515 is T, nucleotide 908 is G, and nucleotide 1001 is C). A reduction in FAD2, including absence of detectable activity, can be achieved by mutagenesis. Decreased FAD2 activity can be inferred from the decreased level of linoleic acid (product) and increased level of oleic acid (substrate) in the plant compared with a corresponding control plant. In one example, the fad2 mutation is a G to A mutation at nucleotide 316 within the fad2-D gene (QTL4A, e.g., SEQ I D NO: QTL4A.n), which results in the substitution of a lysine residue for glutamic acid in a HECGH motif. Such mutations are found within the variety IMC 129, which has been deposited with the ATCC under Accession No. 4081 1. In another example, the fad2 mutation is a T to A mutation at nucleotide 51 5 of the fad2-F gene (e.g., SEQ ID
NO:QTL4B.n), which results in the substitution of a histidine residue for leucine in a
YLNNP motif (amino acid 172 of the Fad2 F polypeptide) (e.g., SEQ ID NO: QTL4B.p). Such a mutation is found within the variety Q508. See U.S. Patent No. 6,342,658. In another example, the fad2 mutation is a G to A mutation at nucleotide 908 of the fad2-F gene (QTL4B), which results in the substitution of a glutamic acid for glycine in the
DRDYG1 LNKV motif (amino acid 303 of the Fad2 F polypeptide). Such a mutation is found within the variety Q4275, which has been deposited with the ATCC under Accession No. 97569. See U.S. Patent No. 6,342,658. Another example of a suitable/cr^ mutation can be the C to T mutation at nucleotide 1001 of the fad2-F gene (as numbered from the ATG) (SEQ ID ON: QTL4B.n, which results in the substitution of an isoleucine for threonine (amino acid 334 of the Fad2 F polypeptide). Such a mutation is found within the high oleic acid variety Q741 5.
In certain embodiments, the modified fad2 loci comprise one or more nucleic acid sequence selected from the group consisting of SEQ ID NO: QTL4A.n wherein nucleotide 316 is A, SEQ ID NO: QTLB.n wherein nucleotide 515 is A, SEQ ID NO: QTL4B.n wherein nucleotide 908 is A, and SEQ ID NO: QTL4B.n wherein nucleotide 1001 is T. In certain embodiments, when the modified fad2 loci comprise a QTL4B locus, the QTL4B locus may comprises a nucleic acid sequence of SEQ ID NO: QTL4B.n comprising one, two or three of the mutations selected from the group consisting of T to A mutation at nucleotide 51 5, G to A mutation at nucleotide 908, and C to T mutation at nucleotide 1001 . Typically, the presence of one of the fad2-D (QTL4A) or fad2-F (QTL4B) mutations confers an oleic acid phenotype wherein the seed oil thereof has about 70% to about 80% oleic acid, while the presence of both fad2-D and fad2-F mutations confers an oleic acid phenotype wherein the seed oil thereof has more than about 80% oleic acid, even without QTL 1 mutation. For example, Q4275 contains the fad2-D mutation from IMC 129 and a fad2-F mutation at amino acid 303. Q508 contains fad2-D mutation from IMC 129 and a fad2-F mutation at amino acid 1 72. Q7415 contains the fad2-D mutation from IMC 129 and a fad2-F mutation at amino acid 334. The presence of both fad2 mutations in Q4275, Q508, and Q7415 confers an oleic acid phenotype of greater than about 80% oleic acid.
5.2.2 Delta-15 desaturase (FAD3)
Brassica plants also can exhibit reduced activity of delta- 1 5 desaturase (FAD3), which is involved in the enzymatic conversion of linoleic acid to a-linolenic acid. The gene encoding FAD3 is referred to as fad3 in Brassica and Arabidopsis. Sequences of higher plant fad3 genes are disclosed in Yadav et al., Plant Physiol.. 103 :467-476 ( 1993), WO 93/1 1245, and Arondel et al., Science, 258: 1353- 1355 (1992). Decreased activity, including absence of detectable activity, of delta-15 desaturase can be achieved by mutagenesis. Decreased activity, including absence of detectable activity, can be inferred from the decreased level of linolenic acid (product) and in some cases, increased level of linoleic acid (the substrate) in the plant compared with a corresponding control plant (e.g. , the Brassica line Topas, ATCC deposit 40624). For example, parent plants can contain the mutation from the APOLLO or STELLAR B. napus variety that confers low linolenic acid. The STELLAR and APOLLO varieties were developed at the University of Manitoba (Manitoba, Canada). In some embodiments, the parents contain the fad3A and/or fad3B mutation from IMC02 that confer a low linolenic acid phenotype. IMC02 contains a mutation in both the fad3A and fad3B genes. In one example, the mutation at the fad3 locus (e.g., SEQ ID NO: QTL6A.n) (the fad3A gene) comprises a C to T mutation at position 2565, numbered from the ATG in genomic DNA, resulting in the substitution of a cysteine for arginine at position 275 of the encoded FAD3A polypeptide. In another example, the mutation at the fad3 locus (e.g., SEQ I D NO: QTL6B.n) (fad3B gene) contains a G to A mutation at position 3053 from ATG in genomic DNA, located in the exon-intron splice site recognition sequence. In another example, the mutation at the fad3 locus (e.g., SEQ ID NO: QTL6C.n) (fad3E gene) contains a G to A mutation at position 1756 from ATG in genomic DNA, located in the exon-intron splice site recognition sequence. In another example, the mutation at the fad3 locus (e.g., SEQ ID NO: QTL6D.n) (fad3d gene) contains a 259 bp deletion that removes 165 bp from the l sl exon. 1MC02 was obtained from a cross of IMCOl x Westar. See Example 3 of U.S. Patent No. 5,750,827. IMCO l was deposited with the American Type Culture Collection (ATCC) under Accession No. 40579. IMC02 was deposited with the ATCC under Accession No. PTA- 6221.
5.3 Beta-ketoacyl-(acyl-carrier-protein) synthase III (kasIII)
Brassica plants also can exhibit reduced activity of KASII I or FabH (β-ketoacyl-ACP synthase III), which is involved in the enzymatic synthesis of C 16 and C26 fatty acids. The gene encoding FAD3 is referred to as kasIII in Brassica. Decreased activity, including absence of detectable activity, of KASII I can be achieved by mutagenesis. Decreased activity, including absence of detectable activity, can be inferred from the decreased level of saturated fatty acids (e.g. C 16 and/or C26 fatty acids) in the plant compared with a corresponding control plant (e.g. the Brassica line Topas, ATCC deposit 40624).
In one embodiment, the Brassica plant disclosed herein comprises nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to a modified kasIII locus (e.g., SEQ ID NO: QTL7.n). One example of QTL7 was identified on chromosome N 19 at position 1284751 24 the genomic sequence of B. napus line Salomon, ATCC deposit designation PTA- 1 1453, as described in WO 201 1 /075716, which is herein incorporated by reference. In one embodiment, the mutation identified in kasIII (SEQ ID NO: QTL7.n) relative to the
Australian cultivar Surpass 400 (Li et al„ 2003; wild type, SEQ ID NO: QTL7.n) is a transition from a "G" in the wild type to an "A" in Salomon at nucleotide 1250, SEQ ID NO: QTL7.n.
Brassica plants comprising the modified kasIII locus produces low amounts of saturated fatty acids in its seed oi l. More speci fically, the amino acid sequence encoded by the wi ld type comprises a glycine at position 252, whereas the KASI II of Salomon (i.e., encoded by the modified kasIII locus SEQ I D NO: QTL7.n) comprises a glutamic acid at that position (SEQ ID NO: QTL7.p).
5.4 Locus on the chromosome Nl (QTL8) related to decreased total saturate content
In some embodiments, the Brassica plants provided herein comprise one or more mutations at the QTL8 locus. In certain of these embodiments, the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences within the segments defined by the chromosome N l (QTL8) SNP markers at positions 20772548 and 22780181 (e.g., between 20843387 and 21080816, or between 20874571 and 20979545) of the genomic sequence of B. napus Salomon line, ATCC deposit designation PTA- 1 1453, as described in WO 201 1 /075716, which is herein incorporated by reference, and wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
In certain embodiments, the Brassica plants comprise one, two, three, four, five, ten, fifteen or more QTL8 markers selected from the group consisting of:
20772548, 20780679, 20843387, 20874199, 20874571 , 20924967, 20979545, 21000713, 21057761 , 21080816, 21 126589, 21 175577, 21244175, 21 273898, 21301 953, 21342623, 21378815, 214253 10, 21491979, and 21549878.
In certain embodiments, the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences within the segments between any two chromosome N l (QTL8) SNP markers at positions selected from: 20772548, 20780679, 20843387, 208741 99, 20874571 , 20924967, 20979545, 21000713, 21057761 , 21080816, 21 126589, 21 1 75577, 212441 75, 21273898, 21301 953, 21 342623, 21 37881 5, 21425310, 21491979, 21 549878, 21597845, 21621627, 21648874, 21700869, 2174091 3, 21793927, 21825553, 21 856527, 21899956, 21938801 , 21980398, 22001 149, 2206051 5, 22100267, 2214431 1 , 22180149, 22217506, 22258914, 22260507, 22299725, 22347689, 22347689, 22379370, 22420077, 22456310, 22498876, 22543 194, 22580394, 22621466, 2265933 1 , 22702378, 22739470, and 22780181 of the genomic sequence of B. napus line Salomon, ATCC deposit designation PTA- 1 1453, wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
In certain embodiments, the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences comprising any one or more (e.g., two, three, or four) chromosome Nl (QTL8) SNP markers at positions selected from: 20772548, 20780679, 20843387, 20874199, 20874571, 20924967, 20979545, 21000713, 21057761, 21080816, 21126589, 21175577, 21244175, 21273898, 21301953, 21342623, 21378815, 21425310, 21491979, 21549878, 21597845, 21621627, 21648874, 21700869, 21740913, 21793927, 21825553, 21856527, 21899956, 21938801, 21980398, 22001149, 22060515, 22100267, 22144311, 22180149, 22217506, 22258914, 22260507, 22299725, 22347689, 22347689, 22379370, 22420077, 22456310, 22498876, 22543194, 22580394, 22621466, 22659331 , 22702378, 22739470, and 22780181 of the genomic sequence of B. napus line Salomon, ATCC deposit designation PTA-11453, wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides. In certain embodiments, the nucleic acid comprises one or more sequences selected from the group consisting of SEQ ID NOs: 1-53, 1-54, 1-55, 1-56, 1-57, 1-58, 1-59, 1-60, 1-61, 1-62, 1-63, 1-64, 1-65, 1-66, 1-67, 1-68, 1-69, 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80, 1-81, 1-82, 1-83, 1-84, 1-85, 1-86, 1-87, 1-88, 1-89, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96, 1-97, 1-98, 1-99, 1-100, 1-101, 1-102, 1-103, and 1- 104.
In certain embodiments, the Brassica plants comprise a nucleic acid sequence having greater than 80% identity to all or part of the genomic sequences between the chromosome Nl (QTL8) SNP markers at positions 20772548 and 22780181 of the B. napus Salomon line, wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
In certain embodiments, the Brassica plants comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve single nucleotide polymorphisms on B. napus chromosome N 1 selected from the group consisting of C to T transitions identified in the B. napus Salomon line, ATCC deposit designation PTA-11453, at locations 20772548,
20780679, 20843387, 20874199, 20874571, 20924967, 20979545, 21000713, 21057761, 21080816, 21126589, and 21175577.
In certain embodiments, the Brassica plants comprise one, two, three, four, five, six, seven, or eight single nucleotide polymorphisms on B. napus chromosome Nl selected from the group consisting of C to T transitions at locations 20772548, 20780679, 20843387, 20874199, 20874571, 20924967, 20979545, and 21000713.
In certain embodiments, the Brassica plant comprises a B. napus N 1 chromosome having greater than 95%, 97.5%, 98%, 99%, 99.9%, 99.99%, 99.999% identity or having 100% identity to all or part of the genomic sequences of the B. napus Salomon line, ATCC deposit designation PTA- 1 1453, on an N l chromosomal segment selected from the group consisting of segments:
beginning with location 20772548 and ending with location 227801 81 or 21342623; beginning with location 20772548 and ending with location 21 1 75577 or. 21 126589; beginning with location 20772548 and ending with location 21080816 or 21057761 ; beginning with location 20772548 and ending with location 21000713 or 20979545; beginning with location 20772548 and ending with location 20924967 or 20874571 ; beginning with location 20780679 and ending with location 22780181 or 21 342623; beginning with location 20780679 and ending with location 21 175577 or 21 126589; beginning with location 20780679 and ending with location 21 080816 or 21057761 ; beginning with location 20780679 and ending with location 2100071 3 or 20979545; beginning with location 20843387 and ending with location 21080816 or 20979545; beginning with location 20924967 and ending with location 21244175 or 21342623; beginning with location 20924967 and ending with location 21 175577 or 21 126589; beginning with location 20924967 and ending with location 21080816 or 21057761 ; and
beginning with location 20924967 and ending with location 21000713 or 20979545; wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
Examples of selected SNPs on chromosome N l (QTL8) are listed below in Table A.
Table A: B. napus position relative to the DH 12075 reference genome, wild-type allele, Salomon allele, flanking sequence and sequence ID number of SNPs identified in
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
5.5 Locus on the chromosome N19 (QTL9) related to decreased total saturate content
In some embodiments, the Brassica plants provided herein comprise one or more mutations at the QTL9 locus. In certain of these embodiments, the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences within the segments defined by: the chromosome N 19 SNP markers at positions 1 1538807 and 18172630 (e.g., 12010676 and 13207412, 12378335 and 12979251 ) of the genomic sequence of B. napus line Salomon, ATCC deposit designation PTA-1 1453, wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
In certain embodiments, the Brassica plants comprise one, two, three, four, five, ten, fifteen or more QTL9 markers selected from the group consisting of:
1 1538807, 1 1763228, 1 1 855685, 12010676, 12205222, 12219881 , 12355162, 12378335,
12507143, 1261 5691 , 12847514, 12979251 , 13003942, 13008581 , 13207412, 133641 32,
13429175, 13429687, 13460532, 13475876, 13504886, and 13704881 .
In certain embodiments, the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences within the segments between any two chromosome Nl 9 (QTL9) SNP markers at positions selected from: 11538807, 11763228, 11855685, 12010676, 12205222, 12219881, 12355162, 12378335, 12507143, 12615691, 12847514, 12979251, 13003942, 13008581, 13207412, 13364132, 13429175, 13429687, 13460532, 13475876, 13504886, 13704881, 13925427, 14046125, 14135213, 14377562, 14776751, 14801661, 15173478, 15235513, 15387929, 15399385, 15547466, 15623646, 15629066, 15684032, 15741164, 15768411, 15898184, 15943625, 15988083, 16211916, 16238183, 16293509, 16468313, 16698792, 16765722, 16787306, 17041989, 17052864, 17111885, 17219357, 17443797, 17636667, 17893475, 17924151, 18164787, and 18172630 of the genomic sequence of B. napus line Salomon, ATCC deposit designation PTA-11453, wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
In certain embodiments, the Brassica plants comprise a nucleic acid having greater than about 80% (e.g., about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.5%, about 99.9%, about 99.995%, or about 99.999%) identity to all or part of the genomic sequences comprising any one or more (e.g., two, three, or four) chromosome Nl 9 (QTL9) SNP markers at positions selected from: 11538807, 11763228, 11855685, 12010676, 12205222, 12219881, 12355162, 12378335, 12507143, 12615691, 12847514, 12979251, 13003942, 13008581, 13207412, 13364132, 13429175, 13429687, 13460532, 13475876, 13504886, 13704881, 13925427, 14046125, 14135213, 14377562, 14776751, 14801661, 15173478, 15235513, 15387929, 15399385, 15547466, 15623646, 15629066, 15684032, 15741164, 15768411, 15898184, 15943625, 15988083, 16211916, 16238183, 16293509, 16468313, 16698792, 16765722, 16787306, 17041989, 17052864, 17111885, 17219357, 17443797, 17636667, 17893475, 17924151, 18164787, and 18172630 of the genomic sequence of B. napus line Salomon, ATCC deposit designation PTA-11453, wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides. In certain embodiments, the nucleic acid comprises one or more sequences selected from the group consistingof SEQ IDNOs: 1-105, 1-106, 1-107, 1-108, 1-109, 1-110, 1-111, 1-112, 1-113, 1- 114, 1-115, 1-116, 1-117, 1-118, 1-119, 1-120, 1-121, 1-122, 1-123, 1-124, 1-125, 1-126, 1- 127, 1-128, 1-129, 1-130, 1-131, 1-132, 1-133, 1-134, 1-135, 1-136, 1-137, 1-138, 1-139, 1- 140, 1-141, 1-142, 1-143, 1-144, 1-145, 1-146, 1-147, 1-148, 1-149, 1-150, 1-151, 1-152, 1- 153, 1-154, 1-155, 1-156, 1-157, 1-158, 1-159, 1-160, 1-161, and 1-162. In certain embodiments, the Brassica plants comprise a nucleic acid sequence having greater than 80% identity to r all or part of the genomic sequence between chromosome N 19 (QTL9) SNP markers at positions 1 1 538807 and 1 8172630 of the B. napus Salomon line, wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
In certain embodiments, the Brassica plants comprise comprising one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen single nucleotide polymorphisms on B. napus chromosome N 19 selected from the group consisting of C to T transitions identified in the B. napus Salomon line, ATCC deposit designation PTA- 1 1453, at locations 1 1 538807, 1 1 763228, 1 1 855685, 12010676, 12205222, 1221 9881 , 12355162, 12378335, 12507143, 1 261 5691 , 12847514, 12979251 , 13003942, 1 3207412, 13364132, 13429175, 13429687, 13460532, 13475876, 13504886, 13704881 , and a G to A transition at location 13008581 .
In certain embodiments, the Brassica plants comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, single nucleotide polymorphisms on B. napus chromosome N 1 9 selected from the group consisting of C to T transitions identified in the B. napus Salomon line, ATCC deposit designation PTA- 1 1453, at locations 1 1 538807, 1 1 763228, 1 1855685, 12010676, 12205222, 12219881 , 12355162, 12378335, 12507143, 1261 5691 , 12847514, 12979251 , 1 3003942, and a G to A transition at location 1 3008581 .
In certain embodiments, the Brassica plants comprise a B. napus N 19 chromosome having greater than 95%, 97.5%, 98%, 99%, 99.9%, 99.99%, 99.999% identity or having 100% identity to all or part of the genomic sequences of the B. napus Salomon line, ATCC deposit designation PTA- 1 1453, on an N 19 chromosomal segment selected from the group consisting of segments:
beginning with location 1 1 538807 and ending with location 1 3704881 or 1 3008581 ; beginning with location 12010676 and ending with location 1 81 72630 or 1 5988083 ; beginning with location 12010676 and ending with location 1 3704881 or 1 3460532; beginning with location 12010676 and ending with location 1 3364132 or 1 3003942; beginning with location 12010676 and ending with location 12979251 or 12847514; beginning with location 12010676 and ending with location 12615691 or 12355162; beginning with location 12010676 and ending with location 1221988 l or 12205222; beginning with location 12355162 and ending with location 12979251 or 13008581 ; beginning with location 12507143 and ending with location 18172630 or 1 5988083; beginning with location 12507143 and ending with location 13704881 or 13460532; beginning with location 12507143 and ending with location 13364132 or 13003942; beginning with location 12507143 and ending with location 12979251 or 12847514; beginning with location 12507143 and ending with location 12615691 or 12355162; and
beginning with location 12507143 and ending with location 1221988 I or 12205222; wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
Examples of selected SNPs on chromosome N l 9 (QTL9) are listed below in Table B. Table B: B. napus position relative to the DH12075 reference genome, wild-type allele, Salomon allele, flanking sequence and sequence ID number of SNPs identified in
QTL9 on N 19.
Figure imgf000039_0001
38
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
6.0 Plants comprising QTL1 and a combination of one or more mutant alleles resulting in reduced activities of fatty acyl-ACP thioesterases (FATA2, and/or FATB), desaturases (FAD2 and/or FAD3), QTL8, and/or QTL9
6.1 Plants comprising QTL1 and one or more mutant alleles at fatB loci, fatA2 locus, fad2 loci, or fad3 loci
In some embodiments, the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus line I MC 106RR and further comprising one or more mutant alleles at one or more fatB loci.
Two or more e.g. , three or four) different mutant alleles at one or more fatB loci (QTL5A, QTL5B, QTL5C, and QTL5D) may be combined in a plant by making a genetic cross between mutant lines. For example, a plant having a mutant allele at a fatB locus (QTL5A) encoding isoform 1 can be crossed or mated with a second plant having a mutant allele at a fatB locus (QTL5B) encoding isoform 2. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds can be screened in order to identify those seeds carrying both mutant alleles. In some embodiments, progeny are selected over multiple generations {e.g. , 2 to 5 generations) to obtain plants having mutant alleles at two different fatB loci. Similarly, a plant having mutant alleles at two or more different FATB isoforms can be crossed with a second plant having mutant alleles at two or more different fatB alleles, and progeny seeds can be screened to identify those seeds carrying mutant alleles at four or more different fatB loci. Again, progeny can be selected for multiple generations to obtain the desired plant.
In some embodiments, the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus line IMC 106RR and further comprising one or more mutant al leles at one or more of the two fatA2 locus.
Mutant alleles at two different fatA2 locus (QTL3A) may be combined in a plant by making a genetic cross between mutant lines, similar to the method described supra.
In some embodiments, the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus line IMC 106RR and further comprising one or more mutant alleles at one or more of the four fad2 loci.
Mutant alleles at two different fad2 loci (QTL4A and QTL4Bmay be combined in a plant by making a genetic cross between mutant lines, simi lar to the method described supra. In some embodiments, the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus line 1MC 106RR and further comprising one or more mutant alleles at three or more fad3 loci.
Mutant alleles at three or four different fad3 loci (QTL6A, QTL6B, QTL6C, and QTL6D) may be combined in a plant by making a genetic cross between mutant lines, simi lar to the method described supra.
6.2 Plants comprising QTL1 and exhibit reduced activity of two or three enzymes selected from FATA2, and FATB; or further exhibit reduced activity of FAD2
6.2.1 Plants comprising QTL1 and exhibit reduced activity of FATA2, and/or
FATB
In some embodiments, the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus line 1MC 106RR and further comprising one or more mutant alleles at one fatA2 locus (QTL3A), and one or more mutant alleles at one, two or more {e.g. , three, or four) of the four different fatB loci (QTL5A, QTL5B, QTL5C, and QTL5D). For example, a plant having a mutant allele at a fatA2 locus can be crossed or mated with a second plant having mutant alleles at two or more different fatB loci. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds can be screened in order to identify those seeds carrying mutant fatA2 and fatB alleles. Progeny can be selected over multiple generations (e.g. , 2 to 5 generations) to obtain plants having a mutant allele at a fatA2 locus and mutant alleles at two or more different fatB loci. As described herein, plants having a mutant al lele at a fatA2b locus and mutant alleles at three or four different fatB loci have a low total saturated fatty acid content that is stable over different growing conditions, i.e. , is less subject to variation due to warmer or colder temperatures during the growing season. Due to the differing substrate profiles of the FatB and FatA enzymes with respect to 16:0 and 18:0, respectively, plants having mutations in fatA2 and fatB loci may exhibit a substantial reduction in amounts of both 16:0 and 1 8:0 in seed oil.
In some embodiments, the Brassica plants described herein are homozygous or heterozygous for the QTL 1 of the B. napus line 1MC 106RR and further comprising one or more mutant alleles at one or more mutant alleles at one, two or more {e.g., three or four) of the four different/o/5 loci (QTL5A, QTL5B, QTL5C, and QTL5D).
In some embodiments, the Brassica plants described herein that are homozygous or heterozygous for the QTL1 of the B. napus I C 106RR and further comprise one or more mutant alleles at one or more mutant alleles at one fatA2 locus, and one or more mutant alleles at one, two, three, or four of the four different fatB loci.
6.2.2 Plants comprising QTLl and exhibit reduced activity of FAD2 and FATA2 and/or FATB
In some embodiments the Brassica plants described herein exhibit reduced activity of
FAD2 in combination with reduced activity of FATA2 and/or FATB.
In one embodiment, the Brassica plants described herein are homozygous or heterozygous for IMC 106RR QTL l and further comprise one or more mutant alleles at one or more (e.g. two, three, or four) of the four different fad2 locus (e.g. , QTL4A and QTL4B) and one or more mutant alleles at:
(i) one fatA2 locus (e.g. , QTL3A) and/or one, two or more (e.g., three or four) of the four different fatB loci (QTL5A, QTL5B, QTL5C, and QTL5D).
6.3 Plants comprising QTLl and exhibit reduced activity of FAD3, and one, two or three enzymes selected from FATA2, and FATB; and/ or further exhibit reduced activity of FAD2.
In one embodiment, the Brassica plants are homozygous or heterozygous for IMC 106RR QTL l and further comprise one or more mutant alleles at three, four, five, or six of the six different fad3 loci (QTL6A, QTL6B, QTL6C, and QTL6D), and one or more mutant alleles at:
(i) one fatA2 locus (e.g. , QTL3 A) and/or one, two or more (e.g., three or four) of the four different/o/5 loci (QTL5A, QTL5B, QTL5C, and QTL5D).
In one embodiment, the Brassica plants homozygous or heterozygous for IMC 106RR QTL l and further comprise one or more mutant alleles at three, or four of the four different fad3 loci (QTL6A, QTL6B, QTL6C, and QTL6D), one or more (e.g. two, three, or four) of the four different/oi/2 loci (e.g. , QTL4A and QTL4B), and:
(i) one fatA2 locus (e.g. , QTL3Aand/or one, two or more (e.g., three or four) of the four different/a/5 loci (QTL5A, QTL5B, QTL5C, and QTL5D).
6.4 Plants comprising QTLl and (1) QTL7 and/or QTL8 and/or QTL9, and/or further exhibit reduced activity of (2) one, two or three enzymes selected from FATA2, and FATB, and/ or (3) FAD2 and/or FAD3
In other embodiments, Brassica plants that are homozygous or heterozygous for QTL l of the B. napus line IMC 106RR also comprise one or more mutant alleles at the locus on a kasIII locus (QTL 7) and/or chromosome N 1 (QTL8) and/or the locus on the chromosome N 19 (QTL9), as described supra.
In another embodiment, Brassica plants that are homozygous or heterozygous for QTL 1 of the B. napus line IMC 106RR also comprise one or more mutant alleles at:
( 1 ) one kasIII locus (QTL 7) and/or the locus on the chromosome N 1 (QTL8) and/or the locus on the chromosome N 19 (QTL9); and
(2) three, or four of the four different fad3 loci (QTL6A, QTL6B, QTL6C, and QTL6D), and/or one or two different fad2 loci (e.g. , QTL4A and QTL4B); and/or one or more mutant alleles at:
(3-i) one fatA2 locus {e.g. , QTL3A) and/or one, two or more {e.g., three or four) of the four different fatB loci (QTL5A, QTL5B, QTL5C, and QTL5D).
Any of the plants described herein that are homozygous or heterozygous for QTL 1 of IMC 106RR may be homozygous or heterozygous for one or more mutant alleles at one, two, three, four, five, six, seven, eight, nine, or ten different loci selected from the group consisting of, QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL7, QTL8, and QTL9. In another embodiment, Brassica plants comprising QTL 1 further comprise ( 1 ) one or more mutant alleles at three or four of different loci selected from the group consisting of QTL6A, QTL6B, QTL6C, and QTL6D; and/or (2) one or more modified {e.g. , mutant) alleles at one, two, three, four, five, six, seven, eight, nine or ten different loci selected from the group consisting of QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL7, QTL8, and QTL9. In certain embodiments, the mutant alleles at loci QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL7, QTL8, and QTL9 are the alleles recited in Table C.
The plants described herein may be modified and/or selected to display a herbicide tolerance trait. That trait can be introduced by selection with the herbicide for which tolerance is sought, or by transgenic means where the genetic basis for the tolerance has been identified. Accordingly, the plants described herein, or parts thereof such as cel ls or protoplasts, may display tolerance to a herbicide selected from the group consisting of imidazolinone, dicamba, cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, L-phosphinothricin, triazine and benzonitrile. Where the plants have been genetically modified to acquire herbicide tolerance by transgenic means they may be non- transgenic to the extent of all other traits except herbicide tolerance. 7.0 Production of Hybrid Brassica Varieties
Hybrid Brassica varieties can be produced by preventing self-pollination of female parent plants {i.e., seed parents), permitting pollen from male parent plants to fertilize such female parent plants, and allowing F| hybrid seeds to form on the female plants. Self- pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development. Alternatively, pollen formation can be prevented on the female parent plants using a form of male steri lity. For example, male sterility can be cytoplasmic male sterility (CMS), nuclear male sterility, molecular male sterility wherein a transgene inhibits microsporogenesis and/or pollen formation, or be produced by self-incompatibility. Female parent plants containing CMS are particularly useful. CMS can be, for example, of the ogu (Ogura), ηαρ, ροί, tour, or mur type. See, for example, Pellan-Delourme and Renard, 1987, and Pellan-Delourme and Renard, 1988. See Riungu and McVetty, 2003 for a description of ηαρ, ροΐ, tour, and mur type CMS.
In embodiments in which the female parent plants are CMS, the male parent plants typically contain a fertility restorer gene to ensure that the F| hybrids are fertile. For example, when the female parent contains an Ogura type CMS, a male parent is used that contains a fertility restorer gene that can overcome the Ogura type CMS. Non-limiting examples of such fertility restorer genes include the Kosena type fertility restorer gene (U.S. Patent No. 5,644,066) and Ogura fertility restorer genes (U.S. Patent Nos. 6,229,072 and 6,392, 127). In other embodiments in which the female parents are CMS, male parents can be used that do not contain a fertility restorer. F| hybrids produced from such parents are male sterile. Male sterile hybrid seed can be inter-planted with male fertile seed to provide pollen for seed-set on the resulting male sterile plants.
The methods described herein can be used to form single-cross Brassica F| hybrids. In such embodiments, the parent plants can be grown as substantially homogeneous adjoining populations to facilitate natural cross-pollination from the male parent plants to the female parent plants. The Fi seed formed on the female parent plants is selectively harvested by conventional means. One also can grow the two parent plants in bulk and harvest a blend of F| hybrid seed formed on the female parent and seed formed upon the male parent as the result of self-pollination. Alternatively, three-way crosses can be carried out wherein a single-cross F| hybrid is used as a female parent and is crossed with a different male parent that satisfies the fatty acid parameters for the female parent of the first cross. Here, assuming a bulk planting, the overall oleic acid content of the vegetable oil may be reduced over that of a single-cross hybrid; however, the seed yield will be further enhanced in view of the good agronomic performance of both parents when making the second cross. As another alternative, double-cross hybrids can be created wherein the Fi progeny of two different single-crosses are themselves crossed. Self-incompatibility can be used to particular advantage to prevent self-pollination of female parents when forming a double-cross hybrid.
Hybrids described herein have good agronomic properties and exhibit hybrid vigor, which results in seed yields that exceed that of either parent used in the formation of the Fi hybrid. For example, yield can be at least 10% (e.g., 10% to about 20%, 10% to about 1 5%, about 1 5% to about 20%, or about 25% to about 35%) above that of either one or both parents. In some embodiments, the yield exceeds that of open-pollinated spring canola varieties such as 46A65 (Pioneer) or Q2 (University of Alberta), when grown under similar growing conditions. For example, yield can be at least 10% (e.g. , 10% to about 1 5% or about 15% to about 20%) above that of an open-pollinated variety.
Hybrids described herein typically produce seed oil having very low levels of glucosinolates (less than30 μτηοl/gram of de-fatted meal at a moisture content of 8.5%). In particular, hybrids can produce seed oil having less than 20 μπιοΐ of glucosinolates/gram of de-fatted meal. As such, hybrids can incorporate mutations that confer low glucosinolate levels. See, for example, U.S. Patent No. 5,866,762. Glucosinolate levels can be determined in accordance with known techniques, including high performance liquid chromatography (HPLC), as described in ISO 9167- 1 : 1992(E), for quantification of total, intact
glucosinolates, and gas-liquid chromatography for quantification of trimethy Isilyl (TMS) derivatives of extracted and purified desulfoglucosinolates. Both the HPLC and TMS methods for determining glucosinolate levels analyze de-fatted or oil-free meal.
8.0 Canola Oil
Brassica plants disclosed herein are useful for producing canola oils with low or no total saturated fatty acids. For example, seed oil of Brassica plants described herein may have a total saturated fatty acid content of about 2.5% to about 5.5%, about 3% to about 5%, about 3% to about 4.5%, about 3.25% to about 3.75%, about 3% to about 3.5%, about 3.4% to about 3.7%, about 3.6% to about 5%, about 4% to about 5.5%, about 4% to about 5%, or about 4.25% to about 5.25%. In some embodiments, an oil has a total saturated fatty acid content of about 4% to about 5.5%, an oleic acid content of about 60% to about 70% (e.g. , about 62% to about 68%, about 63% to about 67%, or about 65% to about 66%), and an a- linolenic acid content of about 2.5% to about 5%. In some embodiments, an oil has a total saturated fatty acid content of about 2.5% to about 5.5% (e.g. , about 4% to about 5%), an oleic acid content of about 71 % to about 80% (e.g. , about 72% to about 78%, about 73% to about 75%, about 74% to about 78%, or about 75% to about 80%) and an a-linolenic acid content of about 2% to about 5.0% (e.g., about 2% to about 2.8%, about 2.25% to about 3%, about 2.5% to about 3%, about 3% to about 3.5%, about 3.25% to about 3.75%, about 3.5% to about 4%, about 3.75% to about 4.25%, about 4% to about 4.5%, about 4.25% to about 4.75%, about 4.5% to about 5%). In some embodiments, a canola oil can have a total saturated fatty acid content of about 2.5% to about 5.5%, an oleic acid content of about 78% to about 80%, and an a-linolenic acid content of no more than about 4% (e.g. , about 2% to about 4%). In some embodiments, an oil has a total saturated fatty acid content of about 3.5% to about 5.5% (e.g., about 4% to about 5%), an oleic acid content of about 81 % to about 90% (e.g. , about 82% to about 88% or about 83% to about 87% oleic acid) and an a-linolenic acid content of about 2% to about 5% (e.g. , about 2% to about 3% or about 3% to about 5%). In some embodiments, an oil has a total saturated fatty acid content of no more than about
3.7% (e.g. , about 3.4% to about 3.7% or about 3.4% to about 3.6%) and an oleic acid content of about 72% to about 75%.
Seed oil of Brassica plants described herein can have a palmitic acid content of about 3.0% to about 5.5% (e.g., about 3.6% to about 5.2%). The stearic acid content of such oils can be about 1.0% to about 3.5% (e.g. , about 1 .3% to about 3.0%). The arachidic acid content of the oil can be about 0.5% to about 1.5% (e.g., about 0.6% to about 1 .1 %). The docosanoic acid content of the oil can be about 0.3% to about 0.8% (e.g. , about 0.4% to about 0.65%).
Seed oil of Brassica plants described herein can have an eicosenoic acid content greater than about 1 .6%, e.g. , about 1 .6% to about 1.9%, about 1 .7% to about 2.3%, about
1 .8% to about 2.3%, or about 1.9% to about 2.3%, in addition to a low total saturates content.
Seed oil of Brassica plants described herein can have a linoleic acid content of about 3% to about 20%, e.g. , about 3.4% to about 5%, about 3.75% to about 5%, about 8% to about 10%, about 10% to about 12%, about 1 1 % to about 1 3%, about 1 3% to about 16%, or about 14% to about 18%, in addition to a low total saturates content.
Seed oil of Brassica plants described herein have an erucic acid content of less than about 2% (e.g., less than about 1 %, about 0.5%, about 0.2%, or about 0.1 %) in addition to a low total saturates content. The fatty acid composition of seed oil of Brassica plants can be determined by first crushing and extracting oil from seed samples (e.g., bulk seed samples of 10 or more seeds). TAGs in the seed are hydrolyzed to produce free fatty acids, which then can be converted to fatty acid methyl esters and analyzed using techniques known to the ski lled artisan, e.g. , gas- liquid chromatography (GLC) according to AOCS Procedure Ce l e-91 . Near infrared (NI R) analysis can be performed on whole seed according to AOCS Procedure Am- 192 (revised 1999)
Seeds harvested from plants described herein can be used to make a crude canola oil or a refined, bleached, and deodorized (RBD) canola oil with a low or no total saturated fatty acid content. Harvested canola seed can be crushed by techniques known in the art. The seed can be tempered by spraying the seed with water to raise the moisture to, for example, about 8.5%. The tempered seed can be flaked using a smooth roller with, for example, a gap setting of 0.23 to 0.27 mm. Heat may be applied to the flakes to deactivate enzymes, facilitate further cell rupturing, coalesce the oil droplets, or agglomerate protein particles in order to ease the extraction process. Typically, oil is removed from the heated canola flakes by a screw press to press out a major fraction of the oil from the flakes. The resulting press cake contains some residual oil.
Crude oil produced from the pressing operation typically is passed through a settling tank with a slotted wire drainage top to remove the solids expressed out with the oil in the screw pressing operation. The clarified oil can be passed through a plate and frame fi lter to remove the remaining fine solid particles. Canola press cake produced from the screw pressing operation can be extracted with commercial n-Hexane. The canola oil recovered from the extraction process is combined with the clarified oil from the screw pressing operation, resulting in a blended crude oil.
Free fatty acids and gums typically are removed from the crude oil by adding food grade phosphoric acid and heating the acidified oil in a batch refining tank. The acid serves to convert the non-hydratable phosphatides to a hydratable form, and to chelate minor metals that are present in the crude oil. The phosphatides and the metal salts are removed from the oil along with the soapstock. The oi l-acid mixture is subsequently treated with sodium hydroxide solution to neutral ize the free fatty acids and the remaining phosphoric acid in the acid-oil mixture. The neutralized free fatty acids, phosphatides, etc. (soapstock) are drained off from the neutralized oil. A water wash may be done to further reduce the soap content of the oil. The oil may be bleached and deodorized before use, if desired, by techniques known in the art.
Oils obtained from the Brassica plant described herein can have increased oxidative stability, which can be measured using, for example, an Oxidative Stability Index Instrument (e.g. , from Omnion, Inc., Rockland, MA) according to AOCS Official Method Cd 12b-92 (revised 1993). Oxidative stability is often expressed in terms of "AOM" hours.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Unless otherwise indicated, all percentages refer to weight % based on total weight % of fatty acids in the oil.
EXAMPLE 1
Brassica plant population SE 1
1.0 Methods
1.1 Phenotyping
Seeds were analyzed for fatty acid profile using gas chromatography. Seeds were crushed, and lipids were extracted using an alkaline extraction method (potassium hydroxide, sodium chloride, iso-octane). The sample was centrifuged and the top layer was used for GC analysis. LSMEANS for each DH line were determined using the GLM procedure in the SAS software package (SAS Institute, 2004).
1.2 Genotyping and QTL Mapping
Genotyping was done using the lllumina (San Diego, CA) Brassica 60K Infinium array at DNA Landmarks (Quebec, Canada). The final list of 1 , 1 79 markers used in linkage map construction was selected based on GenTrain genotype scores above 0.75 as suggested by lllumina followed by selection for those which lack an inter-homoeologous polymorphism (Trick et al., 2009). The genetic linkage map was constructed in JoinMap3 (Van Ooijen and Voorrips, 2001 ) using a threshold recombination frequency of <0.25 and a minimum logarithm of the odds ratio (LOD) score of 6 for grouping loci into linkage groups. The osambi function ( osambi, 1944) was used to calculate genetic distances. Each linkage group was named based on the nomenclature recommended by the Multinational Brassica Genome Project steering committee (http://www.brassica.info/resource/maps/Ig- assignments.php). The map was analyzed further in the R qtl program of the R statistical package (Broman et al., 2003; Broman and Sen, 2009) to confirm marker orders and assess general map quality.
QTL mapping was performed using Haley-Knott Regression (Haley and Knott, 1992) in R/qtl using 1 cM steps. QTL were selected based on significance thresholds made from 1000 permutations (Churchill and Doerge, 1994). Secondary analysis of all polymorphic markers present within the QTL region defined during genome-wide scans were conducted by regressing fatty acid mean values onto molecular marker genotypes (Whittaker, Thompson and Visscher, 1996). For this analysis, markers were aligned in the order of their estimated physical locations.
1.3 Sequencing
High quality DNA was extracted from the fifth leaf of each parent using the standard methods described in the Qiagen (Valencia, CA) column extraction kit. The extracted DNA was run on a 1 % agarose gel to confirm DNA quality and concentrated to 50 ng/μΙ . Parental DNA libraries were sent to the University of Missouri DNA Core Faci lity
(http://biotech.rnet.missouri.edu/dnacore/) and prepped for an average insert size of 200 base pairs. DNA libraries of each parent were sequenced on one lane of an Illumina HiSeq 2000 (San Diego, CA) sequencer to generate 2 x 100 paired-end reads.
1.4 Alignment and polymorphism analysis
Mapping of the genomic sequencing data (fastq files) to a Brassica napus reference genome ( 19 linkage groups of B. napus genotype DH 12075, CanSeq Consortium) was performed using SeqMan NGen v4 (DNAStar, Madison, WI). The alignment was performed using default settings for read mapping and SNP calling. The SNP report created by SeqMan NGen was exported to ArrayStar v4 (DNAStar, Madison, WI) for further filtering. The final list of SNPs was generated using the following filter criteria: quality call score≥ 30 (Phred scale), SNP frequency≥ 5%, depth≥ 5 and "p not ref '≥ 90 (probability that the base is different than the reference base).
2.0 Results
2.1 QTL Mapping
Genome-wide QTL scans discovered one major locus on chromosome N 1 5 that explained a significant proportion of the variation in C 1 8:0, C20:0, C22:0 and total saturates (Table 1 ). Table 1. Summary of QTL location, logarithm of odds (LOD) score, LOD threshold*, percent variance explained (R2), trait mean of lines carrying I C 106RR allele, trait mean of lines carrying Wichita allele and mean differences among lines carrying parental alleles.
Figure imgf000053_0001
* : LOD threshold: alpha = 0.05; based on 1000 permutations.
DH lines carrying the IMC 106RR allele were significantly lower in each fatty acid component than DH lines carrying the Wichita allele. Further analysis of the QTL region using the single marker regression approach found a distinct peak in the R2 value of the marker located at 42,877,3 1 8 bp and a significant improvement in the R2 value relative to the marker located at 42,605, 1 57 bp identified in the original genome-wide scan as the most highly correlated (Table 2).
Table 2. Percent variance explained (R2) of marker genotypes at physical locations* * (42,605, 157 - 43,945,444 bp) for C I 8:0, C20:0, C22:0 and total saturates.
Figure imgf000053_0002
Figure imgf000054_0001
Figure imgf000055_0002
**: Physical locations noted in Table 2 are according to the C genome.
A closer examination of mean trait values of lines carrying alternative alleles at 42,877,318 bp finds a further reduction in mean values of all traits in lines carrying the IMC 106RR allele (Table 3) suggesting this locus may be closer to the causal genetic variant than the original.
Table 3. Trait means and differences among trait means of l ines carrying IMC 106RR and Wichita alleles at 42,877,318 bp (C genome).
Figure imgf000055_0001
Thus, the peak at this location is considered the "center" of the QTL from which we define the QTL interval to encompass a 1 Mb region on either side. The physical locations listed in Table 2 are based on a C genome assembly. The "center" QTL marker (at
42,877,318 bp on C05 of the C genome) is located at 44,730,765 bp on 1 5 of the DH 12075 Brassica napus genome assembly. Therefore, in terms of the B. napus genome, and for the purposes of physical locations for the NGS marker list, the QTL interval is defined as spanning from 43,730,765 - 45,730,765 bp on N l 5 of the DH 12075 B. napus genome assembly.
2.2 Sequencing
Whole-genome sequencing of IMC 106RR and Wichita produced paired-end read data sets summing to 300 million reads (27.8 Gb passed l llumina filter) and 349 mi llion reads (32.2 Gb passed lllumina filter), respectively. The mean Q-score was over 35 with 91 % of reads in each data set considered to be of high-quality (Q-score≥ 30). This equates to an average high-quality read coverage of 23X for I MC 106RR and 26.6X for Wichita for the estimated 1 100 Mbp B. napus genome (Johnston et a!., 2005).
SNP discovery within the defined QTL interval was performed as described. A list of 38 SNPs was generated (Table 4) requiring all selected SNPs to pass all quality thresholds (detailed above), to be homozygous variant and to be unique to the IMC 106RR genotype (i.e., different than the Wichita sequence and the DH 12075 reference sequence).
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
2.3 Candidate genes
Examination of the QTL 1 (N 1 5) interval identified several candidate genes that may be responsible for the observed phenotype (Table 5). The predicted locations of these genes within the QTL 1 (N 1 5) interval are based upon sequence homology with orthologous genes from the well-studied model plant species Arabidopsis thaliana, which is a relative of all Brassica species and is known to have significant gene sequence homology to all Brassica species. These genes were selected as candidate genes based upon their integral activity in plant l ipid biosynthetic pathways.
Table 5. Candidate genes located in the N 1 5 QTL interval.
Figure imgf000062_0001
Figure imgf000063_0001
***: Physical locations are positions according to the DH 12075 B. napus reference genome. OTHER EMBODIMENTS
It is to be understood that, while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
References
All references cited herein and listed below are herein incorporated by reference in their entireties:
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Table C - All possible allele combinations for QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL7, QTL8 AND QTL9:
QTL3A and QTL7, QTL3A and QTL4A, QTL3A and QTL4B, QTL3A and QTL5A, QTL3A and QTL5B, QTL3A and QTL5C, QTL3A and QTL5D, QTL3A and QTL8, QTL3A and QTL9,
QTL7 and QTL4A, QTL7 and QTL4B, QTL7 and QTL5A, QTL7 and QTL5B, QTL7 and QTL5C, QTL7 and QTL5D, QTL7 and QTL8, QTL7 and QTL9,
QTL4A and QTL4B, QTL4A and QTL5A, QTL4A and QTL5B, QTL4A and QTL5C, QTL4A and QTL5D, QTL4A and QTL8, QTL4A and QTL9,
QTL4B and QTL5A, QTL4B and QTL5B, QTL4B and QTL5C, QTL4B and QTL5D, QTL4B and QTL8, QTL4B and QTL9,
QTL5A and QTL5B, QTL5A and QTL5C, QTL5A and QTL5D, QTL5A and QTL8, QTL5A and QTL9,
QTL5B and QTL5C, QTL5B and QTL5D, QTL5B and QTL8, QTL5B and QTL9,
QTL5C and QTL5D, QTL5C and QTL8, QTL5C and QTL9,
QTL5D and QTL8, QTL5D and QTL9,
QTL8 and QTL9,
QTL3A, QTL7 and QTL4A,
QTL3A, QTL7 and QTL4B,
QTL3A, QTL7 and QTL5A,
QTL3A, QTL7 and QTL5B,
QTL3A, QTL7 and QTL5C,
QTL3A, QTL7 and QTL5D,
QTL3A, QTL7 and QTL8,
QTL3A, QTL7 and QTL9,
QTL3A, QTL4A and QTL4B,
QTL3A, QTL4A and QTL5A,
QTL3A, QTL4A and QTL5B,
QTL3A, QTL4A and QTL5C,
QTL3A, QTL4A and QTL5D, QTL3A, QTL4A and QTL8, QTL3A, QTL4A and QTL9, QTL3A, QTL4B and QTL5A, QTL3A, QTL4B and QTL5B, QTL3A, QTL4B and QTL5C, QTL3A, QTL4B and QTL5D, QTL3A, QTL4B and QTL8, QTL3A, QTL4B and QTL9, QTL3A, QTL5A and QTL5B, QTL3A, QTL5A and QTL5C, QTL3A, QTL5A and QTL5D, QTL3A, QTL5A and QTL8, QTL3A, QTL5A and QTL9, QTL3A, QTL5B and QTL5C, QTL3A, QTL5B and QTL5D, QTL3A, QTL5B and QTL8, QTL3A, QTL5B and QTL9, QTL3A, QTL5C and QTL5D, QTL3A, QTL5C and QTL8, QTL3A, QTL5C and QTL9, QTL3A, QTL5D and QTL8, QTL3A, QTL5D and QTL9, QTL3A, QTL8 and QTL9, QTL7, QTL4A and QTL4B, QTL7, QTL4A and QTL5A, QTL7, QTL4A and QTL5B, QTL7, QTL4A and QTL5C, QTL7, QTL4A and QTL5D, QTL7, QTL4A and QTL8, QTL7, QTL4A and QTL9, QTL7, QTL4B and QTL5A, QTL7, QTL4B and QTL5B, QTL7, QTL4B and QTL5C, QTL7, QTL4B and QTL5D, QTL7, QTL4B and QTL8, QTL7, QTL4B and QTL9, QTL7, QTL5A and QTL5B, QTL7, QTL5A and QTL5C, QTL7, QTL5A and QTL5D, QTL7, QTL5A and QTL8, QTL7, QTL5A and QTL9, QTL7, QTL5B and QTL5C, QTL7, QTL5B and QTL5D, QTL7, QTL5B and QTL8, QTL7, QTL5B and QTL9, QTL7, QTL5C and QTL5D, QTL7, QTL5C and QTL8, QTL7, QTL5C and QTL9, QTL7, QTL5D and QTL8, QTL7, QTL5D and QTL9, QTL7, QTL8 and QTL9, QTL4A, QTL4B and QTL5A, QTL4A, QTL4B and QTL5B, QTL4A, QTL4B and QTL5C, QTL4A, QTL4B and QTL5D, QTL4A, QTL4B and QTL8, QTL4A, QTL4B and QTL9, QTL4A, QTL5A and QTL5B, QTL4A, QTL5A and QTL5C, QTL4A, QTL5A and QTL5D, QTL4A, QTL5A and QTL8, QTL4A, QTL5A and QTL9, QTL4A, QTL5B and QTL5C, QTL4A, QTL5B and QTL5D, QTL4A, QTL5B and QTL8, QTL4A, QTL5B and QTL9, QTL4A, QTL5C and QTL5D, QTL4A, QTL5C and QTL8, QTL4A, QTL5C and QTL9, QTL4A, QTL5D and QTL8, QTL4A, QTL5D and QTL9, QTL4A, QTL8 and QTL9, QTL4B, QTL5A and QTL5B, QTL4B, QTL5A and QTL5C, QTL4B, QTL5A and QTL5D, QTL4B, QTL5A and QTL8, QTL4B, QTL5A and QTL9, QTL4B, QTL5B and QTL5C, QTL4B, QTL5B and QTL5D, QTL4B, QTL5B and QTL8, QTL4B, QTL5B and QTL9, QTL4B, QTL5C and QTL5D, QTL4B, QTL5C and QTL8, QTL4B, QTL5C and QTL9, QTL4B, QTL5D and QTL8, QTL4B, QTL5D and QTL9, QTL4B, QTL8 and QTL9, QTL5A, QTL5B and QTL5C, QTL5A, QTL5B and QTL5D, QTL5A, QTL5B and QTL8, QTL5A, QTL5B and QTL9, QTL5A, QTL5C and QTL5D, QTL5A, QTL5C and QTL8, QTL5A, QTL5C and QTL9, QTL5A, QTL5D and QTL8, QTL5A, QTL5D and QTL9, QTL5A, QTL8 and QTL9,
QTL5B, QTL5C and QTL5D, QTL5B, QTL5C and QTL8, QTL5B, QTL5C and QTL9,
QTL5B, QTL5D and QTL8, QTL5B, QTL5D and QTL9, QTL5B, QTL8 and QTL9,
QTL5C, QTL5D and QTL8, QTL5C, QTL5D and QTL9, QTL5C, QTL8 and QTL9,
QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A and QTL4B, QTL3A, QTL7, QTL4A and QTL5A, QTL3A, QTL7, QTL4A and QTL5B, QTL3A, QTL7, QTL4A and QTL5C, QTL3A, QTL7, QTL4A and QTL5D, QTL3A, QTL7, QTL4A and QTL8, QTL3A, QTL7, QTL4A and QTL9, QTL3A, QTL7, QTL4B and QTL5A, QTL3A, QTL7, QTL4B and QTL5B, QTL3A, QTL7, QTL4B and QTL5C, QTL3A, QTL7, QTL4B and QTL5D, QTL3A, QTL7, QTL4B and QTL8, QTL3A, QTL7, QTL4B and QTL9, QTL3A, QTL7, QTL5A and QTL5B, QTL3A, QTL7, QTL5A and QTL5C, QTL3A, QTL7, QTL5A and QTL5D, QTL3A, QTL7, QTL5A and QTL8, QTL3A, QTL7, QTL5A and QTL9, QTL3A, QTL7, QTL5B and QTL5C, QTL3A, QTL7, QTL5B and QTL5D, QTL3A, QTL7, QTL5B and QTL8, QTL3A, QTL7, QTL5B and QTL9, QTL3A, QTL7, QTL5C and QTL5D, QTL3A, QTL7, QTL5C and QTL8, QTL3A, QTL7, QTL5C and QTL9, QTL3A, QTL7, QTL5D and QTL8, QTL3A, QTL7, QTL5D and QTL9, QTL3A, QTL7, QTL8 and QTL9, QTL3A, QTL4A, QTL4B and QTL5A, QTL3A, QTL4A, QTL4B and QTL5B, QTL3A, QTL4A, QTL4B and QTL5C, QTL3A, QTL4A, QTL4B and QTL5D, QTL3A, QTL4A, QTL4B and QTL8, QTL3A, QTL4A, QTL4B and QTL9, QTL3A, QTL4A, QTL5A and QTL5B, QTL3A, QTL4A, QTL5A and QTL5C, QTL3A, QTL4A, QTL5A and QTL5D, QTL3A, QTL4A, QTL5A and QTL8, QTL3A, QTL4A, QTL5A and QTL9, QTL3A, QTL4A, QTL5B and QTL5C, QTL3A, QTL4A, QTL5B and QTL5D, QTL3A, QTL4A, QTL5B and QTL8, QTL3A, QTL4A, QTL5B and QTL9, QTL3A, QTL4A, QTL5C and QTL5D, QTL3A, QTL4A, QTL5C and QTL8, QTL3A, QTL4A, QTL5C and QTL9, QTL3A, QTL4A, QTL5D and QTL8, QTL3A, QTL4A, QTL5D and QTL9, QTL3A, QTL4A, QTL8 and QTL9, QTL3A, QTL4B, QTL5A and QTL5B, QTL3A, QTL4B, QTL5A and QTL5C, QTL3A, QTL4B, QTL5A and QTL5D, QTL3A, QTL4B, QTL5A and QTL8, QTL3A, QTL4B, QTL5A and QTL9, QTL3A, QTL4B, QTL5B and QTL5C, QTL3A, QTL4B, QTL5B and QTL5D, QTL3A, QTL4B, QTL5B and QTL8, QTL3A, QTL4B, QTL5B and QTL9, QTL3A, QTL4B, QTL5C and QTL5D, QTL3A, QTL4B, QTL5C and QTL8, QTL3A, QTL4B, QTL5C and QTL9, QTL3A, QTL4B, QTL5D and QTL8, QTL3A, QTL4B, QTL5D and QTL9, QTL3A, QTL4B, QTL8 and QTL9, QTL3A, QTL5A, QTL5B and QTL5C, QTL3A, QTL5A, QTL5B and QTL5D, QTL3A, QTL5A, QTL5B and QTL8, QTL3A, QTL5A, QTL5B and QTL9, QTL3A, QTL5A, QTL5C and QTL5D, QTL3A, QTL5A, QTL5C and QTL8, QTL3A, QTL5A, QTL5C and QTL9, QTL3A, QTL5A, QTL5D and QTL8, QTL3A, QTL5A, QTL5D and QTL9, QTL3A, QTL5A, QTL8 and QTL9, QTL3A, QTL5B, QTL5C and QTL5D, QTL3A, QTL5B, QTL5C and QTL8, QTL3A, QTL5B, QTL5C and QTL9, QTL3A, QTL5B, QTL5D and QTL8, QTL3A, QTL5B, QTL5D and QTL9, QTL3A, QTL5B, QTL8 and QTL9,
QTL3A, QTL5C, QTL5D and QTL8, QTL3A,QTL5C, QTL5D and QTL9, QTL3A, QTL5C, QTL8 and QTL9, QTL3A, QTL5D, QTL8 and QTL9, QTL7, QTL4A, QTL4B and QTL5A, QTL7, QTL4A, QTL4B and QTL5B, QTL7, QTL4A, QTL4B and QTL5C, QTL7, QTL4A, QTL4B and QTL5D, QTL7, QTL4A, QTL4B and QTL8, QTL7, QTL4A, QTL4B and QTL9, QTL7, QTL4A, QTL5A and QTL5B, QTL7, QTL4A, QTL5A and QTL5C, QTL7, QTL4A, QTL5A and QTL5D, QTL7, QTL4A, QTL5A and QTL8, QTL7, QTL4A, QTL5A and QTL9, QTL7, QTL4A, QTL5B and QTL5C, QTL7, QTL4A, QTL5B and QTL5D, QTL7, QTL4A, QTL5B and QTL8, QTL7, QTL4A, QTL5B and QTL9, QTL7, QTL4A, QTL5C and QTL5D, QTL7, QTL4A, QTL5C and QTL8, QTL7, QTL4A, QTL5C and QTL9, QTL7, QTL4A, QTL5D and QTL8, QTL7, QTL4A, QTL5D and QTL9, QTL7, QTL4A, QTL8 and QTL9, QTL7, QTL4B, QTL5A and QTL5B, QTL7, QTL4B, QTL5A and QTL5C, QTL7, QTL4B, QTL5A and QTL5D, QTL7, QTL4B, QTL5A and QTL8, QTL7, QTL4B, QTL5A and QTL9, QTL7, QTL4B, QTL5B and QTL5C, QTL7, QTL4B, QTL5B and QTL5D, QTL7, QTL4B, QTL5B and QTL8, QTL7, QTL4B, QTL5B and QTL9, QTL7, QTL4B, QTL5C and QTL5D, QTL7, QTL4B, QTL5C and QTL8, QTL7, QTL4B, QTL5C and QTL9, QTL7, QTL4B, QTL5D and QTL8, QTL7, QTL4B, QTL5D and QTL9, QTL7, QTL4B, QTL8 and QTL9, QTL7, QTL5A, QTL5B and QTL5C, QTL7, QTL5A, QTL5B and QTL5D, QTL7, QTL5A, QTL5B and QTL8, QTL7, QTL5A, QTL5B and QTL9, QTL7, QTL5A, QTL5C and QTL5D, QTL7, QTL5A, QTL5C and QTL8, QTL7, QTL5A, QTL5C and QTL9, QTL7, QTL5A, QTL5D and QTL8, QTL7, QTL5A, QTL5D and QTL9, QTL7, QTL5A, QTL8 and QTL9, QTL7, QTL5B, QTL5C and QTL5D, QTL7, QTL5B, QTL5C and QTL8, QTL7, QTL5B, QTL5C and QTL9, QTL7, QTL5B, QTL5D and QTL8, QTL7, QTL5B, QTL5D and QTL9, QTL7, QTL5B, QTL8 and QTL9, QTL7, QTL5C, QTL5D and QTL8, QTL7,QTL5C, QTL5D and QTL9, QTL7, QTL5C, QTL8 and QTL9, QTL7, QTL5D, QTL8 and QTL9, QTL4A, QTL4B, QTL5A and QTL5B, QTL4A, QTL4B, QTL5A and QTL5C, QTL4A, QTL4B, QTL5A and QTL5D, QTL4A, QTL4B, QTL5A and QTL8, QTL4A, QTL4B, QTL5A and QTL9, QTL4A, QTL4B, QTL5B and QTL5C, QTL4A, QTL4B, QTL5B and QTL5D, QTL4A, QTL4B, QTL5B and QTL8, QTL4A, QTL4B, QTL5B and QTL9, QTL4A, QTL4B, QTL5C and QTL5D, QTL4A, QTL4B, QTL5C and QTL8, QTL4A, QTL4B, QTL5C and QTL9, QTL4A, QTL4B, QTL5D and QTL8, QTL4A, QTL4B, QTL5D and QTL9, QTL4A, QTL4B, QTL8 and QTL9, QTL4A, QTL5A, QTL5B and QTL5C, QTL4A, QTL5A, QTL5B and QTL5D, QTL4A, QTL5A, QTL5B and QTL8, QTL4A, QTL5A, QTL5B and QTL9, QTL4A, QTL5A, QTL5C and QTL5D, QTL4A, QTL5A, QTL5C and QTL8, QTL4A, QTL5A, QTL5C and QTL9, QTL4A, QTL5A, QTL5D and QTL8, QTL4A, QTL5A, QTL5D and QTL9, QTL4A, QTL5A, QTL8 and QTL9, QTL4A, QTL5B, QTL5C and QTL5D, QTL4A, QTL5B, QTL5C and QTL8, QTL4A, QTL5B, QTL5C and QTL9, QTL4A, QTL5B, QTL5D and QTL8, QTL4A, QTL5B, QTL5D and QTL9, QTL4A, QTL5B, QTL8 and QTL9, QTL4A, QTL5C, QTL5D and QTL8, QTL4A,QTL5C, QTL5D and QTL9, QTL4A, QTL5C, QTL8 and QTL9, QTL4A, QTL5D, QTL8 and QTL9, QTL4B, QTL5A, QTL5B and QTL5C, QTL4B, QTL5A, QTL5B and QTL5D, QTL4B, QTL5A, QTL5B and QTL8, QTL4B, QTL5A, QTL5B and QTL9, QTL4B, QTL5A, QTL5C and QTL5D, QTL4B, QTL5A, QTL5C and QTL8, QTL4B, QTL5A, QTL5C and QTL9, QTL4B, QTL5A, QTL5D and QTL8, QTL4B, QTL5A, QTL5D and QTL9, QTL4B, QTL5A, QTL8 and QTL9, QTL4B, QTL5B, QTL5C and QTL5D, QTL4B, QTL5B, QTL5C and QTL8, QTL4B, QTL5B, QTL5C and QTL9, QTL4B, QTL5B, QTL5D and QTL8, QTL4B, QTL5B, QTL5D and QTL9, QTL4B, QTL5B, QTL8 and QTL9, QTL4B, QTL5C, QTL5D and QTL8, QTL4B,QTL5C, QTL5D and QTL9, QTL4B, QTL5C, QTL8 and QTL9, QTL4B, QTL5D, QTL8 and QTL9, QTL5A, QTL5B, QTL5C and QTL5D, QTL5A, QTL5B, QTL5C and QTL8, QTL5A, QTL5B, QTL5C and QTL9, QTL5A, QTL5B, QTL5D and QTL8, QTL5A, QTL5B, QTL5D and QTL9, QTL5A, QTL5B, QTL8 and QTL9, QTL5A, QTL5C, QTL5D and QTL8, QTL5A,QTL5C, QTL5D and QTL9,
QTL5A, QTL5C, QTL8 and QTL9,
QTL5A, QTL5D, QTL8 and QTL9,
QTL5B, QTL5C, QTL5D and QTL8, QTL5B.QTL5C, QTL5D and QTL9,
QTL5B, QTL5C, QTL8 and QTL9,
QTL5B, QTL5D, QTL8 and QTL9,
QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B and QTL5A, QTL3A, QTL7, QTL4A, QTL4B and QTL5B, QTL3A, QTL7, QTL4A, QTL4B and QTL5C, QTL3A, QTL7, QTL4A, QTL4B and QTL5D, QTL3A, QTL7, QTL4A, QTL4B and QTL8, QTL3A, QTL7, QTL4A, QTL4B and QTL9, QTL3A, QTL7, QTL4A, QTL5A and QTL5B, QTL3A, QTL7, QTL4A, QTL5A and QTL5C, QTL3A, QTL7, QTL4A, QTL5A and QTL5D, QTL3A, QTL7, QTL4A, QTL5A and QTL8, QTL3A, QTL7, QTL4A, QTL5 A and QTL9, QTL3A, QTL7, QTL4A, QTL5B and QTL5C, QTL3A, QTL7, QTL4A, QTL5B and QTL5D, QTL3A, QTL7, QTL4A, QTL5B and QTL8, QTL3A, QTL7, QTL4A, QTL5B and QTL9, QTL3A, QTL7, QTL4A, QTL5C and QTL5D, QTL3A, QTL7, QTL4A, QTL5C and QTL8, QTL3A, QTL7, QTL4A, QTL5C and QTL9, QTL3A, QTL7, QTL4A, QTL5D and QTL8, QTL3A, QTL7, QTL4A, QTL5D and QTL9, QTL3A, QTL7, QTL4A, QTL8 and QTL9, QTL3A, QTL7, QTL4B, QTL5A and QTL5B, QTL3A, QTL7, QTL4B, QTL5A and QTL5C, QTL3A, QTL7, QTL4B, QTL5A and QTL5D, QTL3A, QTL7, QTL4B, QTL5A and QTL8, QTL3A, QTL7, QTL4B, QTL5A and QTL9, QTL3A, QTL7, QTL4B, QTL5B and QTL5C, QTL3A, QTL7, QTL4B, QTL5B and QTL5D, QTL3A, QTL7, QTL4B, QTL5B and QTL8, QTL3A, QTL7, QTL4B, QTL5B and QTL9, QTL3A, QTL7, QTL4B, QTL5C and QTL5D, QTL3A, QTL7, QTL4B, QTL5C and QTL8, QTL3A, QTL7, QTL4B, QTL5C and QTL9, QTL3A, QTL7, QTL4B, QTL5D and QTL8, QTL3A, QTL7, QTL4B, QTL5D and QTL9, QTL3A, QTL7, QTL4B, QTL8 and QTL9, QTL3A, QTL7, QTL5A, QTL5B and QTL5C, QTL3A, QTL7, QTL5A, QTL5B and QTL5D, QTL3A, QTL7, QTL5A, QTL5B and QTL8, QTL3A, QTL7, QTL5A, QTL5B and QTL9, QTL3A, QTL7, QTL5A, QTL5C and QTL5D, QTL3A, QTL7, QTL5A, QTL5C and QTL8, QTL3A, QTL7, QTL5A, QTL5C and QTL9, QTL3A, QTL7, QTL5A, QTL5D and QTL8, QTL3A, QTL7, QTL5A, QTL5D and QTL9, QTL3A, QTL7, QTL5A, QTL8 and QTL9, QTL3A, QTL7, QTL5B, QTL5C and QTL5D, QTL3A, QTL7, QTL5B, QTL5C and QTL8, QTL3A, QTL7, QTL5B, QTL5C and QTL9, QTL3A, QTL7, QTL5B, QTL5D and QTL8, QTL3A, QTL7, QTL5B, QTL5D and QTL9, QTL3A, QTL7, QTL5B, QTL8 and QTL9, QTL3A, QTL7, QTL5C, QTL5D and QTL8, QTL3A, QTL7,QTL5C, QTL5D and QTL9, QTL3A, QTL7, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A and QTL5B,
QTL3A, QTL4A, QTL4B, QTL5A and QTL5C,
QTL3A, QTL4A, QTL4B, QTL5A and QTL5D,
QTL3A, QTL4A, QTL4B, QTL5A and QTL8,
QTL3A, QTL4A, QTL4B, QTL5A and QTL9,
QTL3A, QTL4A, QTL4B, QTL5B and QTL5C,
QTL3A, QTL4A, QTL4B, QTL5B and QTL5D,
QTL3A, QTL4A, QTL4B, QTL5B and QTL8,
QTL3A, QTL4A, QTL4B, QTL5B and QTL9,
QTL3A, QTL4A, QTL4B, QTL5C and QTL5D,
QTL3A, QTL4A, QTL4B, QTL5C and QTL8,
QTL3A, QTL4A, QTL4B, QTL5C and QTL9,
QTL3A, QTL4A, QTL4B, QTL5D and QTL8,
QTL3A, QTL4A, QTL4B, QTL5D and QTL9,
QTL3A, QTL4A, QTL4B, QTL8 and QTL9,
QTL3A, QTL4A, QTL5A, QTL5B and QTL5C,
QTL3A, QTL4A, QTL5A, QTL5B and QTL5D,
QTL3A, QTL4A, QTL5A, QTL5B and QTL8,
QTL3A, QTL4A, QTL5A, QTL5B and QTL9,
QTL3A, QTL4A, QTL5A, QTL5C and QTL5D,
QTL3A, QTL4A, QTL5A, QTL5C and QTL8,
QTL3A, QTL4A, QTL5A, QTL5C and QTL9,
QTL3A, QTL4A, QTL5A, QTL5D and QTL8,
QTL3A, QTL4A, QTL5A, QTL5D and QTL9,
QTL3A, QTL4A, QTL5A, QTL8 and QTL9,
QTL3A, QTL4A, QTL5B, QTL5C and QTL5D,
QTL3A, QTL4A, QTL5B, QTL5C and QTL8,
QTL3A, QTL4A, QTL5B, QTL5C and QTL9,
QTL3A, QTL4A, QTL5B, QTL5D and QTL8, QTL3A, QTL4A, QTL5B, QTL5D and QTL9,
QTL3A, QTL4A, QTL5B, QTL8 and QTL9,
QTL3A, QTL4A, QTL5C, QTL5D and QTL8,
QTL3A, QTL4A,QTL5C, QTL5D and QTL9,
QTL3A, QTL4A, QTL5C, QTL8 and QTL9,
QTL3A, QTL4A, QTL5D, QTL8 and QTL9,
QTL3A, QTL4B, QTL5A, QTL5B and QTL5C,
QTL3A, QTL4B, QTL5A, QTL5B and QTL5D,
QTL3A, QTL4B, QTL5A, QTL5B and QTL8,
QTL3A, QTL4B, QTL5A, QTL5B and QTL9,
QTL3A, QTL4B, QTL5A, QTL5C and QTL5D,
QTL3A, QTL4B, QTL5A, QTL5C and QTL8,
QTL3A, QTL4B, QTL5A, QTL5C and QTL9,
QTL3A, QTL4B, QTL5A, QTL5D and QTL8,
QTL3A, QTL4B, QTL5A, QTL5D and QTL9,
QTL3A, QTL4B, QTL5A, QTL8 and QTL9,
QTL3A, QTL4B, QTL5B, QTL5C and QTL5D,
QTL3A, QTL4B, QTL5B, QTL5C and QTL8,
QTL3A, QTL4B, QTL5B, QTL5C and QTL9,
QTL3A, QTL4B, QTL5B, QTL5D and QTL8,
QTL3A, QTL4B, QTL5B, QTL5D and QTL9,
QTL3A, QTL4B, QTL5B, QTL8 and QTL9,
QTL3A, QTL4B, QTL5C, QTL5D and QTL8,
QTL3A, QTL4B,QTL5C, QTL5D and QTL9,
QTL3A, QTL4B, QTL5C, QTL8 and QTL9,
QTL3A, QTL4B, QTL5D, QTL8 and QTL9,
QTL3A, QTL5A, QTL5B, QTL5C and QTL5D,
QTL3A, QTL5A, QTL5B, QTL5C and QTL8,
QTL3A, QTL5A, QTL5B, QTL5C and QTL9,
QTL3A, QTL5A, QTL5B, QTL5D and QTL8,
QTL3A, QTL5A, QTL5B, QTL5D and QTL9, QTL3A, QTL5A, QTL5B, QTL8 and QTL9, QTL3A, QTL5A, QTL5C, QTL5D and QTL8, QTL3A, QTL5A,QTL5C, QTL5D and QTL9, QTL3A, QTL5A, QTL5C, QTL8 and QTL9, QTL3A, QTL5A, QTL5D, QTL8 and QTL9, QTL3A, QTL5B, QTL5C, QTL5D and QTL8, QTL3A, QTL5B,QTL5C, QTL5D and QTL9, QTL3A, QTL5B, QTL5C, QTL8 and QTL9, QTL3A, QTL5B, QTL5D, QTL8 and QTL9, QTL3A, QTL5C, QTL5D, QTL8 and QTL9, QTL7, QTL4A, QTL4B, QTL5A and QTL5B, QTL7, QTL4A, QTL4B, QTL5A and QTL5C, QTL7, QTL4A, QTL4B, QTL5A and QTL5D, QTL7, QTL4A, QTL4B, QTL5A and QTL8, QTL7, QTL4A, QTL4B, QTL5A and QTL9, QTL7, QTL4A, QTL4B, QTL5B and QTL5C, QTL7, QTL4A,NQTL4B, QTL5B and QTL5D, QTL7, QTL4A, QTL4B, QTL5B and QTL8, QTL7, QTL4A, QTL4B, QTL5B and QTL9, QTL7, QTL4A, QTL4B, QTL5C and QTL5D, QTL7, QTL4A, QTL4B, QTL5C and QTL8, QTL7, QTL4A, QTL4B, QTL5C and QTL9, QTL7, QTL4A, QTL4B, QTL5D and QTL8, QTL7, QTL4A, QTL4B, QTL5D and QTL9, QTL7, QTL4A, QTL4B, QTL8 and QTL9, QTL7, QTL4A, QTL5A, QTL5B and QTL5C, QTL7, QTL4A, QTL5A, QTL5B and QTL5D, QTL7, QTL4A, QTL5A, QTL5B and QTL8, QTL7, QTL4A, QTL5A, QTL5B and QTL9, QTL7, QTL4A, QTL5A, QTL5C and QTL5D, QTL7, QTL4A, QTL5A, QTL5C and QTL8, QTL7, QTL4A, QTL5A, QTL5C and QTL9, QTL7, QTL4A, QTL5A, QTL5D and QTL8, QTL7, QTL4A, QTL5A, QTL5D and QTL9, QTL7, QTL4A, QTL5A, QTL8 and QTL9, QTL7, QTL4A, QTL5B, QTL5C and QTL5D, QTL7, QTL4A, QTL5B, QTL5C and QTL8, QTL7, QTL4A, QTL5B, QTL5C and QTL9, QTL7, QTL4A, QTL5B, QTL5D and QTL8, QTL7, QTL4A, QTL5B, QTL5D and QTL9, QTL7, QTL4A, QTL5B, QTL8 and QTL9, QTL7, QTL4A, QTL5C, QTL5D and QTL8, QTL7, QTL4A,QTL5C, QTL5D and QTL9, QTL7, QTL4A, QTL5C, QTL8 and QTL9, QTL7, QTL4A, QTL5D, QTL8 and QTL9, QTL7, QTL4B, QTL5A, QTL5B and QTL5C, QTL7, QTL4B, QTL5A, QTL5B and QTL5D, QTL7, QTL4B, QTL5A, QTL5B and QTL8, QTL7, QTL4B, QTL5A, QTL5B and QTL9, QTL7, QTL4B, QTL5A, QTL5C and QTL5D, QTL7, QTL4B, QTL5A, QTL5C and QTL8, QTL7, QTL4B, QTL5A, QTL5C and QTL9, QTL7, QTL4B, QTL5A, QTL5D and QTL8, QTL7, QTL4B, QTL5A, QTL5D and QTL9, QTL7, QTL4B, QTL5A, QTL8 and QTL9, QTL7, QTL4B, QTL5B, QTL5C and QTL5D, QTL7, QTL4B, QTL5B, QTL5C and QTL8, QTL7, QTL4B, QTL5B, QTL5C and QTL9, QTL7, QTL4B, QTL5B, QTL5D and QTL8, QTL7, QTL4B, QTL5B, QTL5D and QTL9, QTL7, QTL4B, QTL5B, QTL8 and QTL9, QTL7, QTL4B, QTL5C, QTL5D and QTL8, QTL7, QTL4B,QTL5C, QTL5D and QTL9, QTL7, QTL4B, QTL5C, QTL8 and QTL9, QTL7, QTL4B, QTL5D, QTL8 and QTL9, QTL7, QTL5A, QTL5B, QTL5C and QTL5D, QTL7, QTL5A, QTL5B, QTL5C and QTL8, QTL7, QTL5A, QTL5B, QTL5C and QTL9, QTL7, QTL5A, QTL5B, QTL5D and QTL8, QTL7, QTL5A, QTL5B, QTL5D and QTL9, QTL7, QTL5A, QTL5B, QTL8 and QTL9, QTL7, QTL5A, QTL5C, QTL5D and QTL8, QTL7, QTL5A,QTL5C, QTL5D and QTL9, QTL7, QTL5A, QTL5C, QTL8 and QTL9, QTL7, QTL5A, QTL5D, QTL8 and QTL9, QTL7, QTL5B, QTL5C, QTL5D and QTL8, QTL7, QTL5B,QTL5C, QTL5D and QTL9, QTL7, QTL5B, QTL5C, QTL8 and QTL9, QTL7, QTL5B, QTL5D, QTL8 and QTL9, QTL7, QTL5C, QTL5D, QTL8 and QTL9, QTL4A, QTL4B, QTL5A, QTL5B and QTL5C, QTL4A, QTL4B, QTL5A, QTL5B and QTL5D, QTL4A, QTL4B, QTL5A, QTL5B and QTL8, QTL4A, QTL4B, QTL5A, QTL5B and QTL9, QTL4A, QTL4B, QTL5A, QTL5C and QTL5D, QTL4A, QTL4B, QTL5A, QTL5C and QTL8, QTL4A, QTL4B, QTL5A, QTL5C and QTL9, QTL4A, QTL4B, QTL5A, QTL5D and QTL8, QTL4A, QTL4B, QTL5A, QTL5D and QTL9, QTL4A, QTL4B, QTL5A, QTL8 and QTL9, QTL4A, QTL4B, QTL5B, QTL5C and QTL5D, QTL4A, QTL4B, QTL5B, QTL5C and QTL8, QTL4A, QTL4B, QTL5B, QTL5C and QTL9, QTL4A, QTL4B, QTL5B, QTL5D and QTL8, QTL4A, QTL4B, QTL5B, QTL5D and QTL9, QTL4A, QTL4B, QTL5B, QTL8 and QTL9, QTL4A, QTL4B, QTL5C, QTL5D and QTL8, QTL4A, QTL4B,QTL5C, QTL5D and QTL9, QTL4A, QTL4B, QTL5C, QTL8 and QTL9, QTL4A, QTL4B, QTL5D, QTL8 and QTL9, QTL4A, QTL5A, QTL5B, QTL5C and QTL5D, QTL4A, QTL5A, QTL5B, QTL5C and QTL8, QTL4A, QTL5A, QTL5B, QTL5C and QTL9, QTL4A, QTL5A, QTL5B, QTL5D and QTL8, QTL4A, QTL5A, QTL5B, QTL5D and QTL9, QTL4A, QTL5A, QTL5B, QTL8 and QTL9, QTL4A, QTL5A, QTL5C, QTL5D and QTL8, QTL4A, QTL5A,QTL5C, QTL5D and QTL9, QTL4A, QTL5A, QTL5C, QTL8 and QTL9, QTL4A, QTL5A, QTL5D, QTL8 and QTL9, QTL4A, QTL5B, QTL5C, QTL5D and QTL8, QTL4A, QTL5B,QTL5C, QTL5D and QTL9, QTL4A, QTL5B, QTL5C, QTL8 and QTL9, QTL4A, QTL5B, QTL5D, QTL8 and QTL9, QTL4A, QTL5C, QTL5D, QTL8 and QTL9, QTL4B, QTL5A, QTL5B, QTL5C and QTL5D, QTL4B, QTL5A, QTL5B, QTL5C and QTL8, QTL4B, QTL5A, QTL5B, QTL5C and QTL9, QTL4B, QTL5A, QTL5B, QTL5D and QTL8, QTL4B, QTL5A, QTL5B, QTL5D and QTL9, QTL4B, QTL5A, QTL5B, QTL8 and QTL9, QTL4B, QTL5A, QTL5C, QTL5D and QTL8, QTL4B, QTL5A,QTL5C, QTL5D and QTL9, QTL4B, QTL5A, QTL5C, QTL8 and QTL9, QTL4B, QTL5A, QTL5D, QTL8 and QTL9,
QTL4B, QTL5B, QTL5C, QTL5D and QTL8,
QTL4B, QTL5B,QTL5C, QTL5D and QTL9,
QTL4B, QTL5B, QTL5C, QTL8 and QTL9,
QTL4B, QTL5B, QTL5D, QTL8 and QTL9,
QTL4B, QTL5C, QTL5D, QTL8 and QTL9,
QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A and QTL5B,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A and QTL5C,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A and QTL5D,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A and QTL8,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B and QTL5C,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B and QTL5D,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B and QTL8,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5C and QTL5D,
QTL3A, QTL7, QTL4A, QTL4B, QTL5C and QTL8,
QTL3A, QTL7, QTL4A, QTL4B, QTL5C and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL4B. QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B and QTL5C,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B and QTL5D,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B and QTL8,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B and QTL9, QTL3A, QTL7, QTL4A, QTL5A, QTL5C and QTL5D,
QTL3A, QTL7, QTL4A, QTL5A, QTL5C and QTL8,
QTL3A, QTL7, QTL4A, QTL5A, QTL5C and QTL9,
QTL3A, QTL7, QTL4A, QTL5A, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL5A, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL5A, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5B, QTL5C and QTL5D,
QTL3A, QTL7, QTL4A, QTL5B, QTL5C and QTL8,
QTL3A, QTL7, QTL4A, QTL5B, QTL5C and QTL9,
QTL3A, QTL7, QTL4A, QTL5B, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL5B, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL5B, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL4A,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B and QTL5C,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B and QTL5D,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B and QTL8,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5C and QTL5D,
QTL3A, QTL7, QTL4B, QTL5A, QTL5C and QTL8,
QTL3A, QTL7, QTL4B, QTL5A, QTL5C and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5D and QTL8,
QTL3A, QTL7, QTL4B, QTL5A, QTL5D and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5B, QTL5C and QTL5D,
QTL3A, QTL7, QTL4B, QTL5B, QTL5C and QTL8,
QTL3A, QTL7, QTL4B, QTL5B, QTL5C and QTL9,
QTL3A, QTL7, QTL4B, QTL5B, QTL5D and QTL8,
QTL3A, QTL7, QTL4B, QTL5B, QTL5D and QTL9, QTL3A, QTL7, QTL4B, QTL5B, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL4B,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL4B, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL5A, QTL5B, QTL5C and QTL5D,
QTL3A, QTL7, QTL5A, QTL5B, QTL5C and QTL8,
QTL3A, QTL7, QTL5A, QTL5B, QTL5C and QTL9,
QTL3A, QTL7, QTL5A, QTL5B, QTL5D and QTL8,
QTL3A, QTL7, QTL5A, QTL5B, QTL5D and QTL9,
QTL3A, QTL7, QTL5A, QTL5B, QTL8 and QTL9,
QTL3A, QTL7, QTL5A, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL5A,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL5A, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL5A, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5B and QTL5C,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5B and QTL5D,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5B and QTL8,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5B and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5C and QTL5D,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5C and QTL8,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5C and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5D and QTL8,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5D and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5B, QTL5C and QTL5D, QTL3A, QTL4A, QTL4B, QTL5B, QTL5C and QTL8,
QTL3A, QTL4A, QTL4B, QTL5B, QTL5C and QTL9,
QTL3A, QTL4A, QTL4B, QTL5B, QTL5D and QTL8,
QTL3A, QTL4A, QTL4B, QTL5B, QTL5D and QTL9,
QTL3A, QTL4A, QTL4B, QTL5B, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5C, QTL5D and QTL8,
QTL3A, QTL4A, QTL4B,QTL5C, QTL5D and QTL9,
QTL3A, QTL4A, QTL4B, QTL5C, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL5A, QTL5B, QTL5C and QTL5D,
QTL3A, QTL4A, QTL5A, QTL5B, QTL5C and QTL8,
QTL3A, QTL4A, QTL5A, QTL5B, QTL5C and QTL9,
QTL3A, QTL4A, QTL5A, QTL5B, QTL5D and QTL8,
QTL3A, QTL4A, QTL5A, QTL5B, QTL5D and QTL9,
QTL3A, QTL4A, QTL5A, QTL5B, QTL8 and QTL9,
QTL3A, QTL4A, QTL5A, QTL5C, QTL5D and QTL8,
QTL3A, QTL4A, QTL5A,QTL5C, QTL5D and QTL9,
QTL3A, QTL4A, QTL5A, QTL5C, QTL8 and QTL9,
QTL3A, QTL4A, QTL5A, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL4A, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL4A, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL4A, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL4B, QTL5A, QTL5B, QTL5C and QTL5D,
QTL3A, QTL4B, QTL5A, QTL5B, QTL5C and QTL8,
QTL3A, QTL4B, QTL5A, QTL5B, QTL5C and QTL9,
QTL3A, QTL4B, QTL5A, QTL5B, QTL5D and QTL8,
QTL3A, QTL4B, QTL5A, QTL5B, QTL5D and QTL9,
QTL3A, QTL4B, QTL5A, QTL5B, QTL8 and QTL9,
QTL3A, QTL4B, QTL5A, QTL5C, QTL5D and QTL8, QTL3A, QTL4B, QTL5A,QTL5C, QTL5D and QTL9,
QTL3A, QTL4B, QTL5A, QTL5C, QTL8 and QTL9,
QTL3A, QTL4B, QTL5A, QTL5D, QTL8 and QTL9,
QTL3A, QTL4B, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL4B, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL4B, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL4B, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL4B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B and QTL5C,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B and QTL5D,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B and QTL8,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5C and QTL5D,
QTL7, QTL4A, QTL4B, QTL5A, QTL5C and QTL8,
QTL7, QTL4A, QTL4B, QTL5A, QTL5C and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5D and QTL8,
QTL7, QTL4A, QTL4B, QTL5A, QTL5D and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5B, QTL5C and QTL5D,
QTL7, QTL4A, QTL4B, QTL5B, QTL5C and QTL8,
QTL7, QTL4A, QTL4B, QTL5B, QTL5C and QTL9,
QTL7, QTL4A, QTL4B, QTL5B, QTL5D and QTL8,
QTL7, QTL4A, QTL4B, QTL5B, QTL5D and QTL9,
QTL7, QTL4A, QTL4B, QTL5B, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5C, QTL5D and QTL8, QTL7, QTL4A, QTL4B,QTL5C, QTL5D and QTL9,
QTL7, QTL4A, QTL4B, QTL5C, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL5A, QTL5B, QTL5C and QTL5D,
QTL7, QTL4A, QTL5A, QTL5B, QTL5C and QTL8,
QTL7, QTL4A, QTL5A, QTL5B, QTL5C and QTL9,
QTL7, QTL4A, QTL5A, QTL5B, QTL5D and QTL8,
QTL7, QTL4A, QTL5A, QTL5B, QTL5D and QTL9,
QTL7, QTL4A, QTL5A, QTL5B, QTL8 and QTL9,
QTL7, QTL4A, QTL5A, QTL5C, QTL5D and QTL8,
QTL7, QTL4A, QTL5A,QTL5C, QTL5D and QTL9,
QTL7, QTL4A, QTL5A, QTL5C, QTL8 and QTL9,
QTL7, QTL4A, QTL5A, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL5B, QTL5C, QTL5D and QTL8,
QTL7, QTL4A, QTL5B,QTL5C, QTL5D and QTL9,
QTL7, QTL4A, QTL5B, QTL5C, QTL8 and QTL9,
QTL7, QTL4A, QTL5B, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL4B, QTL5A, QTL5B, QTL5C and QTL5D,
QTL7, QTL4B, QTL5A, QTL5B, QTL5C and QTL8,
QTL7, QTL4B, QTL5A, QTL5B, QTL5C and QTL9,
QTL7, QTL4B, QTL5A, QTL5B, QTL5D and QTL8,
QTL7, QTL4B, QTL5A, QTL5B, QTL5D and QTL9,
QTL7, QTL4B, QTL5A, QTL5B, QTL8 and QTL9,
QTL7, QTL4B, QTL5A, QTL5C, QTL5D and QTL8,
QTL7, QTL4B, QTL5A,QTL5C, QTL5D and QTL9,
QTL7, QTL4B, QTL5A, QTL5C, QTL8 and QTL9,
QTL7, QTL4B, QTL5A, QTL5D, QTL8 and QTL9,
QTL7, QTL4B, QTL5B, QTL5C, QTL5D and QTL8,
QTL7, QTL4B, QTL5B,QTL5C, QTL5D and QTL9,
QTL7, QTL4B, QTL5B, QTL5C, QTL8 and QTL9, QTL7, QTL4B, QTL5B, QTL5D, QTL8 and QTL9,
QTL7, QTL4B, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL7, QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL7, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL7, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL7, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL4A, QTL4B, QTL5A, QTL5B, QTL5C and QTL5D,
QTL4A, QTL4B, QTL5A, QTL5B, QTL5C and QTL8,
QTL4A, QTL4B, QTL5A, QTL5B, QTL5C and QTL9,
QTL4A, QTL4B, QTL5A, QTL5B, QTL5D and QTL8,
QTL4A, QTL4B, QTL5A, QTL5B, QTL5D and QTL9,
QTL4A, QTL4B, QTL5A, QTL5B, QTL8 and QTL9,
QTL4A, QTL4B, QTL5A, QTL5C, QTL5D and QTL8,
QTL4A, QTL4B, QTL5A,QTL5C, QTL5D and QTL9,
QTL4A, QTL4B, QTL5A, QTL5C, QTL8 and QTL9,
QTL4A, QTL4B, QTL5A, QTL5D, QTL8 and QTL9,
QTL4A, QTL4B, QTL5B, QTL5C, QTL5D and QTL8,
QTL4A, QTL4B, QTL5B,QTL5C, QTL5D and QTL9,
QTL4A, QTL4B, QTL5B, QTL5C, QTL8 and QTL9,
QTL4A, QTL4B, QTL5B, QTL5D, QTL8 and QTL9,
QTL4A, QTL4B, QTL5C, QTL5D, QTL8 and QTL9,
QTL4A, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL4A, QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL4A, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL4A, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL4A, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL4A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL4B, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL4B, QTL5A, QTL5B,QTL5C, QTL5D and QTL9, QTL4B, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL4B, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL4B, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL4B, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B and QTL5C,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B and QTL5D,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B and QTL8,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5C and QTL5D,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5C and QTL8,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5C and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B, QTL5C and QTL5D,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B, QTL5C and QTL8,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B, QTL5C and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL4B,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B, QTL5C and QTL5D,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B, QTL5C and QTL8,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B, QTL5C and QTL9,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B, QTL8 and QTL9, QTL3A, QTL7, QTL4A, QTL5A, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL5A,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL5A, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5A, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B, QTL5C and QTL5D,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B, QTL5C and QTL8,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B, QTL5C and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B, QTL5D and QTL8,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B, QTL5D and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL4B, QTL5A,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL4B, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL4B, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C and QTL5D, QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C and QTL8,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5D and QTL8,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5D and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5C, QTL5D and QTL8,
QTL3A, QTL4A, QTL4B, QTL5A,QTL5C, QTL5D and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5C, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL4A, QTL4B, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL4A, QTL4B, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL4A, QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL4A, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL4A, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL4B, QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL4B, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL4B, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL4B, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL4B, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C and QTL5D,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C and QTL8,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5D and QTL8, QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5D and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5C, QTL5D and QTL8,
QTL7, QTL4A, QTL4B, QTL5A,QTL5C, QTL5D and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5C, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5B, QTL5C, QTL5D and QTL8,
QTL7, QTL4A, QTL4B, QTL5B,QTL5C, QTL5D and QTL9,
QTL7, QTL4A, QTL4B, QTL5B, QTL5C, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5B, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL7, QTL4A, QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL7, QTL4A, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL7, QTL4A, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL7, QTL4B, QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL7, QTL4B, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL7, QTL4B, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL7, QTL4B, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL4B, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL4A, QTL4B, QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL4A, QTL4B, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL4A, QTL4B, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL4A, QTL4B, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL4A, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C and QTL5D,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C and QTL8,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5B,QTL5C, QTL5D and QTL9, QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL4B, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL4A, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D and QTL8,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B,QTL5C, QTL5D and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9,
QTL3A, QTL7, QTL4A, QTL4B, QTL5A, QTL5B, QTL5C, QTL5D, QTL8 and QTL9.
Table D
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001

Claims

Claims Claims
1. A Brassica plant, or a part thereof, that is non-transgenic or that is free of transgenes other than those for herbicide tolerance, the Brassica plant comprising a nucleic acid sequence having greater than 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.9%, 99.99% or 99.999% identity to all or part of the genomic sequences between positions 43730765 and 45730765 of chromosome Nl 5 (QTL1) of the B. napus line IMC 106RR (National Registration No. 5118), wherein said part of the genomic sequences of the B. napus IMC106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides, and wherein said Brassica plant further comprises a mutant allele at one or more genetic loci selected from the group consisting of:
(i) fatA2 locus,
(ii) fad2 loci,
(iii) fatB loci,
(iv) kasIII locus,
(v) QTL8 locus, and
(vi) QTL9 locus.
2. A Brassica plant, or a part thereof, that is non-transgenic or that is free of transgenes other than those for herbicide tolerance, the Brassica plant comprising a nucleic acid sequence having greater than 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.9%, 99.99% or 99.999% identity to all of, or a part comprising greater than 20, 30, 40, 50 or 60 contiguous nucleotides of, the genomic sequences between positions 43730765 and 45730765 of the chromosome N15 (QTLl) ofthe 5. napus line IMC106RR (National Registration No. 5118),
wherein said Brassica plant further comprises one or more mutant alleles at:
(i) one or more fatA2 locus and/or fatB loci.
3. The Brassica plant, or a part thereof, according to claim 2, further comprises one or more mutant alleles at one or more fad2 loci.
4. A Brassica plant, or a part thereof, that is non-transgenic or that is free of transgenes other than those for herbicide tolerance, the Brassica plant comprising a nucleic acid sequence having greater than 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.9%, 99.99% or 99.999% identity to all or part of the genomic sequences between positions 43730765 and 45730765 of the chromosome N15 (QTL1) of the B. napus line IMC106RR (National Registration No. 5118), wherein said part of the genomic sequences of the B. napus IMC106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides, and wherein said Brassica plant further comprises one or more mutant alleles at:
(i) one or more fatA2 locus and/or fad2 loci; or
(ii) one or more fatB loci and/or fad2 loci.
5. The Brassica plant, or a part thereof, according to any preceding claim, wherein the Brassica comprises and is homozygous or heterozygous for one or more mutant alleles at:
(i) one or more kasIII locus (QTL7) and/or QTL8 locus (on chromosome Nl);
(ii) one or more QTL8 locus and/or the QTL9 locus (on chromosome N19); or
(iii) one kasIII locus, the QTL8 locus and the QTL9 locus.
6. The Brassica plant, or part thereof , according to claim 5, wherein the mutant allele at QTL8 comprises a nucleic acid having greater than 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.9%, 99.99%) or 99.999%) identity to the genomic sequence of B. napus line Salomon between any two SNP markers on Nl at positions selected from: 20772548, 20780679, 20843387, 20874199, 20874571, 20924967, 20979545, 21000713, 21057761, 21080816, 21126589, 21175577, 21244175, 21273898, 21301953, 21342623, 21378815, 21425310, 21491979, 21549878, 21597845, 21621627, 21648874, 21700869, 21740913, 21793927, 21825553, 21856527, 21899956, 21938801, 21980398, 22001149, 22060515, 22100267, 22144311, 22180149, 22217506, 22258914, 22260507, 22299725, 22347689, 22347689, 22379370, 22420077, 22456310, 22498876, 22543194, 22580394, 22621466, 22659331, 22702378, 22739470, and 22780181, wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides; and/or wherein the mutant allele at QTL9 comprises a nucleic acid having greater than 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.9%, 99.99% or 99.999% identity to the genomic sequence of B. napus line Salomon between any two SNP markers on N19 at positions selected from: 11538807, 11763228, 11855685, 12010676, 12205222, 12219881, 12355162, 12378335, 12507143, 12615691, 12847514, 12979251, 13003942, 13008581, 13207412, 13364132, 13429175, 13429687, 13460532, 13475876, 13504886, 13704881, 13925427, 14046125, 14135213, 14377562, 14776751, 14801661, 15173478, 15235513, 15387929, 15399385, 15547466, 15623646, 15629066, 15684032, 15741164, 15768411, 15898184, 15943625, 15988083, 16211916, 16238183, 16293509, 16468313, 16698792, 16765722, 16787306, 17041989, 17052864, 17111885, 17219357, 17443797, 17636667, 17893475, 17924151, 18164787, and 18172630, and wherein said part of the genomic sequences of the B. napus Salomon line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
7. The Brassica plant, or a part thereof, according to any preceding claim, wherein the genomic sequences between the chromosome N15 (QTL1) SNP markers at positions 43730765 and 45730765 comprises a nucleic acid having greater than 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.9%, 99.99% or 99.999% identity to the genomic sequence of B. napus line
IMC106RR between any two SNP markers at positions selected from: 43849348, 43877053, 44223135, 44252880, 44407747, 44617229, 44659768, 44736857, 44746871, 44756604, 44771673, 44794771, 44796526, 44798855, 44839954, 44855978, 44859545, 44872594, 44923355, 44985743, 45007425, 45093354, 45147723, 45159244, 45165499, 45256038, 45260347, 45278355, 45298286, 45312516, 45354975, 45365977, 45378346, 45402371, 45409080, 45496931, 45499138, and 45720715, and wherein said part of the genomic sequences of the B. napus IMC106RR line comprises greater than 20, 30, 40, 50, or 60 contiguous nucleotides.
9. The Brassica plant, or a part thereof, according to any preceding claim, wherein said Brassica plant comprises and is homozygous or heterozygous for one or more mutant alleles at one or two fat A 2 locus.
10. The Brassica plant, or a part thereof, according to any preceding claim, wherein said Brassica plant comprises and is homozygous or heterozygous for one or more mutant alleles at one, two, three, or four fatB loci.
11. The Brassica plant, or a part thereof, according to any preceding claim, wherein said Brassica plant comprises and is homozygous or heterozygous for one or more mutant alleles at one or two fad2 loci.
12. The Brassica plant, or a part thereof, according to any preceding claim, wherein said Brassica plant comprises and is homozygous or heterozygous for one or more mutant alleles three, or four fad3 loci.
13. The Brassica plant, or a part thereof, according to any preceding claim, wherein said plant is a Brassica napus, Brassica juncea, Brassica oleracea, Brassica carinata or Brassica rapa plant.
14. The Brassica plant, or a part thereof, according to any preceding claim, wherein a seed oil of said plant has a total saturated fatty acid content less than about 2.5%, less than about 2.0%, less than about 1.5%, less than about 1.25%, less than about 1.2%, less than about 1.1%, less than about 1.0%, less than about 0.95%, less than about 0.9%, less than about 0.085%), or less than about 0.8%.
15. The Brassica plant, or a part thereof, according to any preceding claim, wherein said plant produces a seed oil having a total saturates content of about 0.5% to about 2.5%, about 0.5% to about 2.0%), about 0.5% to about 1.5%, about 0.5% to about 1.25%, about 0.5% to about 1.2%, about 0.5% to about 1.1%, about 0.5% to about 1.0%, about 0.5% to about 0.95%, about 0.5% to about 0.9%, about 0.5% to about 0.085%, or about 0.5% to about 0.8%.
16. The Brassica plant, or a part thereof, according to any preceding claim, wherein said plant produces a seed oil having a palmitic acid (C16:0) content of about 3.6% to about 5.2%.
17. The Brassica plant, or a part thereof, according to any preceding claim, wherein said plant produces a seed oil having a stearic acid (C18:0) content of about 1.3% to about 3.0%>.
18. The Brassica plant, or a part thereof, according to any preceding claim, wherein said plant produces a seed oil having an arachidic acid (C20:0) content about 0.60% to about 1.10%.
19. The Brassica plant, or a part thereof, according to any preceding claim, wherein said plant produces a seed oil having a docosanoic acid (C22:0) content of about 0.40% to about 0.65%.
20. Progeny of a plant of any preceding claim, where in the progeny comprises the nucleic acid sequence having greater than 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.9%, 99.99% or 99.999%) identity to the genomic sequences between positions 43730765 and 45730765 of the chromosome N 15 (QTL1) of the B. napus line IMC 106RR (National Registration No. 5118).
21. The progeny plant, or part thereof, according to claim 20, wherein the progeny plant is homozygous or heterozygous for one or more mutant alleles at:
(i) one fatA2 locus;
(ii) one or two fad2 loci;
(iii) three or four fad3 loci;
(iv) one, two, three, or four fatB loci;
(v) the QTL8 locus, and
(vi) the QTL9 locus.
22. The progeny plant, or a part thereof, according to claim 21, wherein said Brassica plant comprises and is homozygous or heterozygous for one or more mutant alleles one or two fad3 loci.
23. A plant or a cell of a plant of any one of claims 1 to 22, wherein said plant has tolerance to a herbicide selected from the group consisting of imidazolinone, dicamba, cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, L-phosphinothricin, triazine and benzonitrile.
24. A seed of a plant according to any of claims 1 to 23.
25. A method of producing an oil, said method comprising extracting oil from at least one seed of claim 24, said oil from said crushed seed, said oil having, after refining, bleaching, and deodorizing, a total saturates content of about 0.5% to about 5.5% or about 0.5% to about 1%.
26. The method of claim 25, wherein said plant produces seeds yielding an oil having a palmitic acid (C16:0) content of about 3.6% to about 5.2%.
27. The method of any of claims 25 to 26, wherein said plant produces seeds yielding an oil having a stearic acid (CI 8:0) content of about 1.3% to about 3.0%.
28. The method of any of claims 25 to 27, wherein said plant produces seeds yielding an oil having an arachidic acid (C20:0) content about 0.60% to about 1.10%.
29. The method of any of claims 25 to 28, wherein said plant produces seeds yielding an oil having a docosanoic acid (C22:0) content of about 0.40% to about 0.65%.
30. A method of DNA-assisted selection of a reduced-palmitic acid oil trait in Brassica plants, comprising: a) assessing the DNA of the Brassica plants to determine the presence or absence of all of, or a part comprising greater than 20, 30, 40, 50 or 60 contiguous nucleotides of, QTLl; and b) selecting one of the Brassica plants in which QTLl, or at least said part of QTLl, is present.
31. The method of claim 30, further comprising selecting a second one of the Brassica plants in which QTLl, or at least said part of QTLl, is present.
32. The method of claim 30, further comprising: a) crossing the selected canola plant with a second canola plant to produce a Fl Brassica plant; and b) assessing the DNA of the Fl Brassica plant to determine the presence or absence of QTLl .
33. The method of claim 31 , further comprising: a) crossing each of the selected Brassica plants with a second canola plant to produce a plurality of Fl Brassica plants b) assessing the DNA of the Fl Brassica plants to determine the presence or absence of QTLl; and c) selecting one of the Fl Brassica plants in which QTLl , or at least said part of QTLl, is present.
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WO2020168277A1 (en) * 2019-02-14 2020-08-20 Cargill, Incorporated Brassica plants producing elevated levels of polyunsaturated fatty acids
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