Classifications

• • 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]
• • 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
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
  • A01H1/10—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits
• • 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
  • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
  • A01H5/10—Seeds
• • 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
• • 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
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/13—Plant traits

Definitions

• sequence listing is submitted concurrently with the specification as an XML formatted sequence listing with a file named 108398-WO-SEC ST.26 created on October 2, 2023 and having a size of 74,739 bytes.
• This sequence listing is part of the specification and is herein incorporated by reference in its entirety.
• This disclosure relates to the discovery of a quantitative trait locus (QTL) associated with a low fiber content trait in canola (Brassica napus , provided are methods and compositions for identifying the low fiber content trait in canola as well as methods for breeding with, selecting, introgressing, and/or introducing the low fiber content QTL trait into canola.
• QTL quantitative trait locus
• Canola meal the fraction of the seed remaining after crushing and oil extraction, is approximately 55% of the volume of canola seed.
• Canola meal consists of several components including protein, fiber, residual oil, carbohydrates, and anti-nutritional factors. Although canola meal is relatively high in protein, its high fiber content decreases its digestibility and its value as an animal feed. Compared to soybean meal, canola meal contains higher values of dietary fiber and a lower percentage of protein. Because of its high dietary fiber, canola meal has about 20% less metabolizable energy (ME) than soybean meal. As a result, the value of the meal has remained low relative to other oilseed meals such as soybean meal, particularly in rations for pigs and poultry. Rakow (2004a) Canola meal quality improvement through the breeding of yellow -seeded varieties — an historical perspective, in AAFC Sustainable Production Systems Bulletin. Additionally, the presence of glucosinolates in some canola meals also decreases its value, due to the deleterious effects these compounds have on the growth and reproduction of livestock.
• ME metabolizable energy
• Canola varieties are distinguished in part by their seed coat color. Seed coat color is generally divided into two main classes: yellow and black (or dark brown). Varying shades of these colors, such as reddish brown and yellowish brown, are also observed. Canola varieties with lighter seed coat color have been widely observed to have thinner hulls, and thus less fiber and more oil and protein than varieties with dark color seed coats. Stringam et al. (1974) Chemical and morphological characteristics associated with seed coat color in rapeseed, in Proceedings of the 4th International Rapeseed Congress, Giessen, Germany, pp. 99-108; Bell and Shires (1982) Can. J.
• the foregoing method can be used for introgression of the N15 deletion QTL disclosed herein. After crossing the first Brassica plant with the second Brassica plant, producing and selecting a progeny plant or germplasm thereof having the N15 deletion QTL as described herein, the selected progeny plant is backcrossed with the second plant (the recurrent parent) lacking the N15 deletion QTL to produce a second generation of progeny plants.
• a plurality of second- generation progeny plants can be screened and at least one second generation progeny plant having the screened-for N15 deletion QTL can be selected in accordance with any method disclosed herein, thereby producing a second-generation progeny plant that combines desirable attributes the recurrent parent with the N15 deletion QTL and its associated low fiber trait disclosed herein.
• This process can be repeated two, three, four, five, six, or seven times, i.e., by crossing the latest generation of selected backcross progeny plants having the N15 deletion QTL with the recurrent parent plant, and each time identifying and selecting additional backcross progeny plants having N15 deletion QTL.
• Repeated backcrossing to the recurrent parent plant can be used to create Brassica plant lines that combine (i) the N15 deletion QTL and (ii) the agronomic characteristics of the recurrent parent plant, when backcross lines and recurrent parent are grown in the same environmental conditions.
• the modification can be done in a Brassica plant, cell, or germplasm that comprises wild-type N15 chromosome (i.e., without the N15 QTL disclosed herein).
• the modification can include creating a deleted segment located between genomic sequences SEQ ID NO:65 (N101HTV-001) and SEQ ID NO:78 (N101HGY-001).
• the deletion can be include deleted genomic DNA between position 86.09 cM and position 101.53 cM of chromosome N15, e.g., between position 97.93 cM and position 101.24 cM of chromosome N15.
• the deleted segment can be at least about 500 kb in length, at least about 532 kb in length, at least about 575 kb in length, at least about 600 kb in length, at least about 700 kb in length, at least about 800 kb in length, at least about 900 kb in length, at least about 1,000 kb in length, or at least about 1,667 kb in length.
• the method can also include inserting a heterologous insertion sequence, i.e., an insertion that has replaced the deleted genomic segment on chromosome N15.
• Methods for creating such gene edited plants dropouts comprise inducing a first and second double strand break in genomic DNA using a TALE-nuclease (TALEN), a meganuclease, a zinc finger nuclease, or a CRISPR-associated nuclease.
• the method comprises introducing a CRISPR-associated nuclease and guide RNAs into a B. napus plant cell.
• ECM enhanced canola meal or
• FIG. 2 is schema showing relative locations of forward primers, reverse primers and probes that can be used to assay the presence, absence, and/or zygosity of the N15 deletion. Top line represents wild-type N15 chromosome, bottom line represents N15 with the deletion discovered in NS7627MC, dashed line represents replacement sequence.
• Primers that can be used to distinguish wild-type andNS7627MC sequences as follows: “1” and “2” indicate forward and revers primers, respectively that amplify wild-type sequence N15 sequence (no deletion); “4 and “5” indicate forward and revers primers, respectively that amplify insertion sequence that has replaced and thereby indicates the presence of N15 deletion. Short lines “3” and “6” indicate probes to detect amplicons produced in wild-type line and N15 deletion line, respectively.
• nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. ⁇ 1.822. Although only one strand of each nucleic acid sequence is shown or referenced, the complementary strand is understood to be included by any reference to the displayed strand.
• Allotetraploid generally refers to a hybrid organism that has a chromosome set that is four times that of a haploid organism.
• An “allele” is one of several alternative forms of a gene occupying a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are the same, that plant is “homozygous” at that locus. If the alleles present at a given locus on a chromosome differ, that plant is “heterozygous” at that locus.
• a “Cas endonuclease” may comprise domains that enable it to function as a double-strand- break-inducing agent.
• a “Cas endonuclease” may also comprise one or more modifications or mutations that abolish or reduce its ability to cleave a double-strand polynucleotide (dCas).
• the Cas endonuclease molecule may retain the ability to nick a single-strand polynucleotide (for example, a D10A mutation in a Cas9 endonuclease molecule) (nCas9).
• the guide polynucleotide/Cas endonuclease complex When complexed with a guide polynucleotide, the guide polynucleotide/Cas endonuclease complex”, (or “guide polynucleotide/Cas endonuclease system”, “ guide polynucleotide/Cas complex”, “guide polynucleotide/Cas system” and “guided Cas system” or “Polynucleotide-guided endonuclease” , “PGEN”” are capable of directing the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and nick or cleave (introduce a single or double-strand break) the DNA target site.
• a guided Cas system referred to herein can comprise Cas protein(s) and suitable polynucleotide component(s) of any known CRISPR systems (Horvath and Barrangou, 2010, Science 327: 167-170; Makarova etal. 2015, Nature Reviews Microbiology Vol. 13:1-15; Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015, Molecular Cell 60, 1- 13).
• elite line means any line that has resulted from breeding and selection for superior agronomic performance.
• An elite plant is any plant from an elite line.
• Enhanced canola meal refers to a meal, which is made by processing Brassica napus seeds and which has decreased fiber content, and may have increased protein and true metabolizable energy content, as well as reduced anti- nutritional factors such as glucosinolates, tannins, phytic acid, sinapine and erucic acid. Meal with some or all of these characteristics could allow increasing inclusion rates in the diet of animal species especially in monogastric animals.
• the enhanced canola meal made from seeds of the N15 deletion Brassica napus oilseed disclosed herein may variously be referred to herein as “ECM” and includes “black seeded canola ECM,” “BSC ECM,” or “dark seeded canola ECM;” the present disclosure is not limited to black-seeded canola and black seeded canola ECM.
• Fiber is a component of plant cell walls, and includes carbohydrate polymers (e.g., cellulose (linear glucose polymeric chains)); hemicellulose (branched chains of heteropolymers of, for example, galactose, xylose, arabinose, rhamnose, with phenolic molecules attached); and pectins (water soluble polymers of galacturonic acid, xylose, arabinose, with different degrees of methylation). Fiber also includes polyphenolic polymers (e.g., lignin-like polymers and condensed tannins).
• carbohydrate polymers e.g., cellulose (linear glucose polymeric chains)
• hemicellulose branched chains of heteropolymers of, for example, galactose, xylose, arabinose, rhamnose, with phenolic molecules attached
• pectins water soluble polymers of galacturonic acid, xylose, arabinose, with different degrees of methylation.
• Fiber also includes
• ADF Acid Detergent Fiber
• NDF Neutral Detergent Fiber
• ADF is a measure of the plant components in forages that is least digestible by livestock, including cellulose and lignin.
• NDF measures most of the structural components in plant cells (i.e. lignin, hemicellulose and cellulose), but not pectin. Decreased ADF and NDF also results in more digestible, higher energy meal.
• genotype of an individual or group of individuals is defined and described by the allele forms at the one or more loci that the individual has inherited from its parents.
• genotype may also be used to refer to an individual's genetic constitution at a single locus, at multiple loci, or at all the loci in its genome.
• a “haplotype” is the genotype of an individual at a plurality of genetic loci.
• the genetic loci described by a haplotype may be physically and genetically linked; i.e., the loci may be positioned on the same chromosome segment.
• germplasm refers to genetic material of or from an individual plant or group of plants (e.g., a plant line, variety, and family), and a clone derived from a plant or group of plants.
• a germplasm may be part of an organism or cell, or it may be separate (e.g., isolated) from the organism or cell.
• germplasm provides genetic material with a specific molecular makeup that is the basis for hereditary qualities of the plant.
• “germplasm” refers to cells of a specific plant; seed; tissue of the specific plant (e.g., tissue from which new plants may be grown); and non-seed parts of the specific plant (e.g., leaf, stem, pollen, and cells).
• germplasm is synonymous with “genetic material,” and it may be used to refer to seed (or other plant material) from which a plant may be propagated.
• a germplasm utilized in a method or plant as described herein is from a. Brassica line or variety.
• a germplasm is seed of the Brassica line or variety.
• a germplasm is a nucleic acid sample from the Brassica line or variety.
• introgression refers to the transmission of an allele at a genetic locus into a genetic background.
• introgression of a specific allele form at the locus may occur by transmitting the allele form to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the specific allele form in its genome.
• Progeny comprising the specific allele form may be repeatedly backcrossed to a line having a desired genetic background. Backcross progeny may be selected for the specific allele form, so as to produce a new variety wherein the specific allele form has been fixed in the genetic background.
• introgression of a specific allele form may occur by recombination between two donor genomes (e.g., in a fused protoplast), where at least one of the donor genomes has the specific allele form in its genome.
• Introgression may involve transmission of a specific allele form that may be, for example and without limitation, a selected allele form of a marker allele; a QTL; and/or a transgene.
• introgression may involve transmission of a disclosed chromosome N15 deletion into a progeny plant.
• linkage between genes or markers may refer to the phenomenon in which genes or markers on a chromosome show a measurable probability of being passed on together to individuals in the next generation. Thus, linkage of one marker to another marker or gene may be measured and/or expressed as a recombination frequency. The closer two genes or markers are to each other, the closer to “1” this probability becomes. Thus, the term “linked” may refer to one or more genes or markers that are passed together with a gene with a probability greater than 0.5 (which is expected from independent assortment where markers/genes are located on different chromosomes).
• markers that are linked to the gene may be said to be linked to the phenotype.
• the term “linked” may refer to a relationship between a marker and a gene, or between a marker and a phenotype.
• a relative genetic distance is generally proportional to the physical distance (measured in base pairs) that two linked markers or genes are separated from each other on a chromosome.
• One centimorgan is defined as the distance between two genetic markers that show a 1% recombination frequency (i.e., a crossing-over event occurs between the two markers once in every 100 cell divisions).
• centimorgan is defined as the distance between two genetic markers that show a 1% recombination frequency (i.e., a crossing-over event occurs between the two markers once in every 100 cell divisions).
• the closer one marker is to another marker or gene whether the distance between them is measured in terms of genetic distance or physical distance
• chromosomal distance is approximately proportional to the frequency of recombination events between traits, there is an approximate physical distance that correlates with recombination frequency.
• the term “tightly linked” may refer to one or more genes or markers that are located within about 35 cM of one another.
• two “tightly linked” genes or markers may be separated by less than 36 cM; less than 35 cM; less than 34 cM; less than about 33 cM; less than about 32 cM; less than about 31 cM; less than about 30 cM; less than about 29 cM; less than about 28 cM; less than about 27 cM; less than about 26 cM; less than about 25 cM; less than about 24 cM; less than about 23 cM; less than about 22 cM; less than about 21 cM; less than about 20 cM; less than about 19 cM; less than about 18 cM; less than about 17 cM; less than about 16 cM; less than about 15 cM; less than about 14 cM; less than about 13 cM; less than about 12 cM; less than about
• the term “extremely tightly-linked” may refer to one or more genes or markers that are located within about 5.0 cM of one another.
• two “extremely tightly -linked” genes or markers may be separated by less than 6.0 cM; less than 5.5 cM; less than 5.0 cM; less than about 4.5 cM; less than about 4.0 cM; less than about 3.5 cM; less than about 3.0 cM; less than about 2.5 cM; less than about 2.0 cM; less than about 1.5 cM; less than about 1.0 cM; and less than about 0.5 cM.
• markers linked to a particular gene or phenotype include those markers that are tightly linked, and those markers that are extremely tightly linked, to the gene or phenotype.
• a “marker allele” or “marker allele form” refers to the version of the marker that is present in a particular individual.
• the term “marker” as used herein may refer to a cloned segment of chromosomal DNA and may also or alternatively refer to a DNA molecule that is complementary to a cloned segment of chromosomal DNA.
• the term also refers to nucleic acid sequences complementary to genomic marker sequences, such as nucleic acid primers and probes.
• a marker may be described, for example, as a specific polymorphic genetic element at a specific location in the genetic map of an organism.
• a genetic map may be a graphical representation of a genome (or a portion of a genome, such as a single chromosome) where the distances between landmarks on the chromosome are measured by the recombination frequencies between the landmarks.
• a genetic landmark can be any of a variety of known polymorphic markers, for example and without limitation: simple sequence repeat (SSR) markers; restriction fragment length polymorphism (RFLP) markers; and single nucleotide polymorphism (SNP) markers.
• SSR markers can be derived from genomic or expressed nucleic acids (e.g., expressed sequence tags (ESTs)).
• Isozymes are multiple forms of enzymes that differ from one another with respect to amino acid sequence (and therefore with respect to their encoding nucleic acid sequences). Some isozymes are multimeric enzymes containing slightly different subunits. Other isozymes are either multimeric or monomeric, but have been cleaved from a pro-enzyme at different sites in the pro-enzyme amino acid sequence. Isozymes may be characterized and analyzed at the protein level or at the nucleic acid level. Thus, any of the nucleic acid-based methods described herein can be used to analyze isozyme markers in particular examples.
• genetic marker alleles that are polymorphic in a population can be detected and distinguished by one or more analytic methods such as, PCR-based sequence specific amplification methods, RFLP analysis, AFLP analysis, isozyme marker analysis, SNP analysis, SSR analysis, allele specific hybridization (ASH) analysis, detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), randomly amplified polymorphic DNA (RAPD) analysis.
• analytic methods such as, PCR-based sequence specific amplification methods, RFLP analysis, AFLP analysis, isozyme marker analysis, SNP analysis, SSR analysis, allele specific hybridization (ASH) analysis, detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), randomly amplified polymorphic DNA (RAPD) analysis.
• SSRs simple sequence repeats
• RAPD randomly amplified polymorphic DNA
• the genetic markers disclosed herein can be used in MAS programs to identity Brassica varieties that have or can generate progeny that have reduced fiber (when compared to parental varieties, siblings and/or otherwise isogenic plants lacking the N15 deletion), to identify individual Brassica plants comprising this reduced fiber QTL, and to breed this QTL into other Brassica varieties to reduce their fiber content.
• a “marker set” or a “set” of markers or probes refers to a specific collection of markers (or data derived therefrom) that may be used to identify individuals comprising a trait of interest.
• a set of markers linked to N15 deletion may be used to identify a Brassica plant comprising one or more copies of an N15 deletion disclosed herein.
• Data corresponding to a marker set may be stored in an electronic medium. While each marker in a marker set may possess utility with respect to trait identification, individual markers selected from the set and subsets including some, but not all, of the markers may also be effective in identifying individuals comprising the trait of interest.
• a “mutated gene” or “modified gene” is a gene that has been altered through human intervention. Such a “mutated” or “modified” gene has a sequence that differs from the sequence of the corresponding non-mutated gene by at least one nucleotide addition, deletion, or substitution.
• the mutated or modified gene comprises an excision or deletion of a sequence of nucleotides within that results from two double strands break which are specifically targeted within a genomic sequence by guide polynucleotide/Cas endonuclease system or a gene edited tool as disclosed herein.
• a “gene edited” or “modified” plant is a plant comprising a mutated gene or deletion.
• a “targeted mutation” is a mutation in a gene (referred to as the target gene), including a native gene, that was made by altering a target sequence within the target gene using any method known to one skilled in the art, including a method involving a guided Cas endonuclease system as disclosed herein.
• nucleic acid molecule may refer to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above.
• a nucleotide may refer to a ribonucleotide, deoxyribonucleotide, or a modified form of either type of nucleotide.
• a “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” The term includes single- and double-stranded forms of DNA.
• a nucleic acid molecule can include either or both naturally occurring and modified nucleotides, linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
• nucleic acid molecule also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations.
• Quantitative trait may refer to a trait or phenotype that is expressed in varying degrees, along a generally continuous gradient and is frequently linked to two or more genes and is affected by environment.
• markers are genetically linked to a QTL (or to another marker) are known to those of skill in the art and include, for example and without limitation, standard linear models (e.g., ANOVA or regression mapping; Haley and Knott (1992) Heredity 69:315); and maximum likelihood methods (e.g., expectation-maximization algorithms; Lander and Botstein (1989) Genetics 121 : 185-99; Jansen (1992) Theor. Appl. Genet. 85:252-60; Jansen (1993) Biometrics 49:227-31; Jansen (1994) “Mapping of quantitative trait loci by using genetic markers: an overview of biometrical models,” In J. W.
• nucleic acid sequencing e.g., next-generation sequencing or NGS
• primer extension e.g., allele-specific PCR (e.g. KASP), H2-dependent PCR (rhPCR), Melt Analysis of Mismatch Amplification Mutation Assay (Melt-MAMA), MasscodeTM (Qiagen, Germantown, Md.), Invader® (Hologic, Madison, Wis.), Serial Invasive Signal Amplification Reaction (SISAR), Snapshot® (Applied Biosystems, Foster City, Calif.), and Taqman® (Applied Biosystems, Foster City, Calif.).
• SNP markers are highly useful, availability of high-quality DNA sequence information is necessary for their discovery.
• Brainssica plant may refer to, for example and without limitation, a whole plant; multiple plants; Brassica plant cell(s); plant protoplast; plant tissue culture (e.g., from which a whole plant can be regenerated); plant callus; plant parts (e.g., seed, flower, cotyledon, leaf, stem, bud, root, and root tip); and plant cells that are intact in a plant or in a part of a plant.
• Plant line As used herein, a “line” refers to a group of plants that display little genetic variation (e.g., no genetic variation) between individuals for at least one trait. Inbred lines may be created by several generations of self-pollination and selection or, alternatively, by vegetative propagation from a single parent using tissue or cell culture techniques. As used herein, the terms “cultivar,” “variety,” and “type” are synonymous, and these terms refer to a line that is used for commercial production.
• a “variety” or “cultivar” is a plant line that is used for commercial production which is distinct, stable and uniform in its characteristics when propagated. In the case of a hybrid variety or cultivar, the parental lines are distinct, stable, and uniform in their characteristics.
• Trait or phenotype The terms “trait” and “phenotype” are used interchangeably herein.
• the traits of particular interest are low fiber content and, in some cases, seed coat color.
• Some canola varieties exhibit a yellow seed coat, while further varieties exhibit a dark (e.g., black, dark, and mottled) seed coat.
• Methods for detecting (identifying) Brassica napus plants or germplasm that carry particular alleles of low fiber content markers are a feature of some embodiments.
• any of a variety of marker detection protocols available in the art may be used to detect a marker allele, depending on the type of marker being detected.
• suitable methods for marker detection may include amplification and identification of the resulting amplified marker by, for example and without limitation, PCR; LCR; and transcription-based amplification methods (e.g., SNP detection, SSR detection, RFLP analysis, and many others).
• Markers corresponding to genetic polymorphisms between members of a population may be detected by any of numerous methods including, for example and without limitation, nucleic acid amplification-based methods; and nucleotide sequencing of a polymorphic marker region.
• Many detection methods including amplification-based and sequencing-based methods
• this disclosure further provides methods of identifying and/or selecting a low fiber content Brassicanapus plant or germplasm, comprising: (a) detecting, in Brassica napus plant or germplasm, the presence of one or more genetic markers associated with low fiber content in a Brassica napus plant, as described herein; and (b) selecting said Brassica napus plant or germplasm based on the presence of the one or more genetic markers associated with low fiber content in a Brassica napus plant.
• the methods disclosed herein include detecting an amplified DNA fragment associated with the presence of a particular allele of a SNP.
• the detecting of a particular allele of a SNP can be performed by any of a number or techniques, including, but not limited to, the use of detectable labels.
• Detectable labels suitable for use include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.
• a particular allele of a SNP may be detected using, for example, autoradiography, fluorography, or other similar detection techniques, depending on the particular label to be detected.
• Useful labels include biotin (for staining with labeled streptavidin conjugate), magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetric labels.
• Other labels include ligands that bind to antibodies or specific binding targets labeled with fluorophores, chemiluminescent agents, and enzymes.
• detection techniques include the use of fluorescent dyes.
• the disclosure provides a method comprising the transfer by introgression of the nucleic acid sequence from a N15 deletion QTL donor Brassica plant into a standard or higher fiber content recipient Brassica plant by crossing the plants. This transfer can be accomplished by using traditional breeding techniques.
• the N15 deletion locus associated with low fiber content can be introgressed into elite or commercial Brassica varieties using marker-assisted selection (MAS) or marker-assisted breeding (MAB).
• MAS and MAB involve the use of one or more of the molecular markers, identified as having a significant likelihood of co-segregation with a desired trait, and used for the identification and selection of those offspring plants that contain one or more of the genes that encode the desired trait.
• such identification and selection are based on the selection of one or more marker sequences located in one of the N15 QTL intervals disclosed herein or one or more markers associated with N15 deletions disclosed herein.
• MAB can also be used to develop near-isogenic lines (NIL) harboring one or more low fiber content alleles of interest, allowing a more detailed study of an effect of such allele(s), and is also an effective method for development of backcross inbred line (BIL) populations.
• Brassica plants developed according to these embodiments can, in some exmples, derive a majority of their traits from the recipient plant and derive the low fiber content trait from the donor plant.
• MAB/MAS techniques increase the efficiency of backcrossing and introgressing genes using marker-assisted selection (MAS) or marker-assisted breeding (MAB).
• inbred low fiber content Brassica plant lines can be developed using the techniques of recurrent selection and backcrossing, selfing, and/or dihaploids, or any other technique used to make parental lines.
• low fiber content can be introgressed into a target recipient plant (the recurrent parent) by crossing the recurrent parent with a first donor plant, which differs from the recurrent parent and is referred to herein as the “non-recurrent parent.”
• the recurrent parent is a plant that has high fiber content and, in some cases, comprises commercially desirable characteristics, such as, but not limited to disease and/or insect resistance, valuable nutritional characteristics, valuable abiotic stress tolerance (including, but not limited to, drought tolerance, salt tolerance), and the like.
• the non-recurrent parent exhibits low fiber content and comprises a nucleic acid sequence that is associated with low fiber content.
• the non-recurrent parent can be any plant variety or inbred line that is cross- fertile with the recurrent parent.
• the progeny resulting from a cross between the recurrent parent and non-recurrent parent are backcrossed to the recurrent parent.
• the resulting plant population is then screened for the desired characteristics, which screening can occur in a number of different ways. For instance, the population can be screened using phenotypic pathology screens or quantitative bioassays as are known in the art.
• the population can be screened using phenotypic pathology screens or quantitative bioassays as are known in the art.
• MAB can be performed using one or more of the herein before described molecular markers to identify those progeny that comprise a nucleic acid sequence associated with low fiber content. Also, MAB can be used to confirm the results obtained from the quantitative bioassays.
• the markers defined herein are suitable to select proper offspring plants by genotypic screening.
• the Fl hybrid plants that exhibit a low fiber content phenotype or, in some embodiments, the genotype, and thus comprise the requisite nucleic acid sequence associated with low fiber content can then be selected and backcrossed to the recurrent parent for one or more generations in order to allow for the Brassica plant to become increasingly inbred. This process can be performed for one, two, three, four, five, six, seven, eight, or more generations.
• sequences and markers disclosed herein which are associated with the N15 deletion QTL can be used in MAS methods to identify and/or select and/or produce progeny having the N15 deletion QTL associated with low fiber content.
• the present disclosure provides a method of selecting a low fiber content Brassica plant, the method comprising: detecting, in a Brassica germplasm, the presence of a sequence or marker associated with the N15 deletion QTL a Brassica plant, wherein said sequence or marker is located within the chromosomal interval disclosed herein, and selecting a plant from said germplasm, thereby selecting a plant having the N15 deletion QTL associated with lower fiber content.
• the disclosed chromosomal interval can be the interval defined by and including SEQ ID NO:65 (N101HTV- 001) and SEQ ID NO:78 (N101HGY-001) and the N15 deletion is associated with low fiber content in Brassica napus.
• the disclosed method can include screening the sample for the absence of one or more genomic markers located between genomic position 86.09 cM and position 101.53 cM of chromosome N15, e.g., between position 97.93 cM and position 101.24 cM of chromosome N15, thereby identifying and/or selecting a low fiber content Brassica napus plant or germplasm.
• Also provided herein is a method of producing a low fiber content plant and/or germplasm, the method comprising: crossing a first canola plant or germplasm with a second canola plant or germplasm, wherein said first canola plant or germplasm comprises within its genome the N15 deletion QTL disclosed herein, collecting seed from the cross and growing a progeny canola plant from the seed, wherein said progeny canola plant comprises in its genome said the N15 deletion QTL associated with low fiber content and can be used to produce a low fiber content canola plant.
• the second canola plant or germplasm used in the method is of an elite variety of canola.
• the crossing of the first and second canola plants produces a progeny canola plant or germplasm having the low fiber content marker introgressed into a genome that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% identical to that of an elite variety of canola.
• the present disclosure provides Brassica plants and germplasms having low fiber content. As discussed above, the disclosed methods can be utilized to identify, select and/or produce a canola plant or germplasm having the disclosed N15 deletion QTL which is associated with low fiber content. In addition to the methods described above, a.
• Brassica plant or germplasm having low fiber content may be produced by any method whereby an N15 deletion disclosed herein is introduced into the Brassica plant or germplasm by such methods that include, but are not limited to, transformation (including, but not limited to, bacterial-mediated nucleic acid delivery (e.g., via Agrobacteria)), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome mediated nucleic acid delivery, microinjection, microparticle bombardment, electroporation, sonication, infiltration, PEG- mediated nucleic acid uptake, as well as any other electrical, chemical, physical (mechanical) and/or biological mechanism that results in the introduction of nucleic acid into the plant cell, or any combination thereof, protoplast transformation or fusion, a double haploid technique, embryo rescue, or by any other nucleic acid transfer system.
• transformation including, but not limited to, bacterial-mediated nucleic acid delivery (e.g., via Agrobacteria)
• nucleic acid molecules in the context of a plant cell, plant and/or plant part means contacting a nucleic acid molecule with the plant, plant part, and/or plant cell in such a manner that the nucleic acid molecule gains access to the interior of the plant cell and/or a cell of the plant and/or plant part.
• these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different nucleic acid constructs.
• these polynucleotides can be introduced into plant cells in a single transformation event, in separate transformation events, or, e.g., as part of a breeding protocol.
• transformation refers to the introduction of a heterologous nucleic acid into a cell.
• a canola plant, or part thereof, having a genetic marker associated with low fiber content obtainable by the methods of the presently disclosed subject matter, are aspects of the presently disclosed subject matter.
• the canola plant or germplasm may be the progeny of a cross between an elite variety of canola and a variety of canola that comprises an allele associated with low fiber content.
• the canola plant or germplasm is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identical to that of an elite variety of canola.
• the canola plant or germplasm may be the progeny of an introgression wherein the recurrent parent is an elite variety of canola and the donor comprises a genetic marker associated (e.g., SNP, combination of SNPs, SNP located in a chromosome interval) with low fiber content in a canola plant as described herein.
• a genetic marker associated e.g., SNP, combination of SNPs, SNP located in a chromosome interval
• the canola plant or germplasm may be the progeny of a cross between a first elite variety of canola (e.g., a tester line) and the progeny of a cross between a second elite variety of canola (e.g., a recurrent parent) and a variety of canola that comprises a genetic marker associated with low fiber content in a canola plant as described herein (e.g., a donor).
• a first elite variety of canola e.g., a tester line
• a second elite variety of canola e.g., a recurrent parent
• a variety of canola that comprises a genetic marker associated with low fiber content in a canola plant as described herein e.g., a donor
• Another aspect of the presently disclosed subject matter relates to a method of producing seeds that can be grown into low fiber content canola plants.
• the method comprises providing a low fiber content canola plant of this invention, crossing the low fiber content canola plant with another canola plant, and collecting seeds resulting from the cross, which when planted, produce low fiber content canola plants.
• the provided herein are improved canola plants, seeds, and/or canola tissue culture produced by the methods described herein.
• the presently disclosed subject matter provides methods for analyzing the genomes of canola plants/germplasms to identify those that include desired markers associated with low fiber content.
• the methods of analysis comprise amplifying subsequences of the genomes of the canola plants/germplasms and determining the nucleotides present in one, some, or all positions of the amplified subsequences.
• the present disclosure provides methods for detecting alleles associated with low fiber content in canola.
• allele discrimination is performed in a microtiter plate using Infmium Bead ChipTM technology and GoldenGateTM allele-specific extension PCR-based assay (Illumina, San Diego, CA), which identifies each SNP with a discrete fluorescent tag and a unique address to target a particular bead in the array.
• the reaction products or fluorescent intensities on the beads are captured and the SNP allele associated with low fiber content in canola is determined.
• Example 1 Plant materials and Identification of an ADF QTL [01231 A DH population was developed from two black seeded elite canola lines.
• NS7627MC is a male spring canola type that provides low fiber content and high protein in the seed meal.
• G00178MC is also a male spring line that provides a commodity oil profile as well as low fiber content and high protein in the seed meal.
• the two lines differed by approximately 3 percent for ADF content, and approximately 2 percent in the protein content in the meal as measured by NIR, results shown in Table 1.
• 181 DH plants were generated and phenotyped using two different approaches. In the first approach, greenhouse (GH) grown seed from this population were subjected to NIR measurements for ADF and protein content in the meal; the same GH seed was also phenotyped using mass spectrometry (MS).
• MS mass spectrometry
• the second phenotyping approach involved growing the same DH population in fields at two separate locations in Manitoba and Saskatoon. Field grown seed was subjected to NIR, and phenotypic data analyzed by Best Linear Unbiased Predictions (BLUP) algorithm to account for spatial, year effects and location variability. BLUP values generated were used as phenotypes for trait mapping.
• the DH population’s 181 individuals were genotyped using 10,800 Illumina XT genotyping assays. Composite interval analyses were performed for QTL detection.
• Two ADF- quantitative trait loci (QTLs) were identified on chromosomes N7 and N15 using greenhouse phenotyping data.
• the ADF-QTL on N15 explained more than 50% of the phenotypic variance for ADF in this population.
• Logarithm-of-odds method indicated that the QTL on N15 is linked to the ADF phenotype with a very high degree of statistical significance.
• a threshold LOD score of at least 3 is typically used to establish high probablility of genetic linkage. In these experiments, an LOD score of 31.6 indicated a very strongly correlation between N15 QTL and low ADF phenotype.
• Example 3 Genomic Characterization and Confirmation of N15 Deletions. Two complimentary approaches were employed to validate the presence of N15 segmental deletion described in Example 2. In one approach, short-read whole genome re-sequencing data was generated via Illumina sequencing platform for two elite lines: NS7627MC and NS8290BC that contain the N15 low fiber locus and the deletion at around 97.93-101.24 cM, as indicated by marker haplotype profile. Short-read sequences from these two lines were aligned to another, using wild-type proprietary reference genome NS1822BC as control, and the alignments were visualized using Integrated Genomics Viewer (IGV) software. Robinson et al. (2011) Nature Biotechnology , 29(1): 24-26.
• IGF Integrated Genomics Viewer
• a segment of about 575 kb in the control NS1822BC genome has no corresponding short-read sequence matches on the N15 chromosome from NS7627MC genome.
• about 1.65 Mb of N15 NS1822BC genome has no corresponding shortsequence match in NS8290BC genome.
• this insertion comprises sequence matches to chromosome N5, while the remaining insertion sequence has high similarity to several other B. napus chromosomes and could not be designated as a duplication from a single chromosome. Since the corresponding N5 homoeologous segment in NS7627MC is intact, it is unlikely that a simple N5 to N15 one-way non-reciprocal translocation occurred. Similarly, the corresponding deletion segment in NS8290BC was replaced by an approximately 1.1 Mb insertion segment. Approximately 580 kb of this second insertion segment includes sequence matches to N5 homoeologous chromosome, while the remaining has high similarity to several other B.
• NS7627MC deletion start point position 101 of SEQ ID NO:80
• NS7627MC deletion end point position 101 of SEQ ID NO:81
• NS8290BC start point position 101 of SEQ ID NO:82
• NS8290BC end point position 101 of SEQ ID NO:83
• Primer and probe sequences were also designed to amplify and detect DNA sequences within the insertion segment (at the N15 deletion locus) in NS7627MC and/or NS8290BC - these are examples of an N15 deletion assay (DEL Assay).
• DEL Assay N15 deletion assay
• probe for detecting wild type N15 segment was tagged with a VIC fluorophore
• the probe for detecting the insertion segment i.e., indicating N15 segment deletion
• NS7627MC DEL Assay was tagged with a FAM fluorophore.

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