CLUBROOT RESISTANCE IN BRASSICA

Abstract

Provided are methods and compositions, including assays, probes and primers for identifying Brassica plants that are resistant to clubroot disease. Also provided are breeding methods for introducing a clubroot resistance phenotype into Brassica plants and/or their progeny.

Claims

1. A method for introducing a clubroot resistance locus into a Brassica plant the method comprising: crossing a first parent Brassica plant comprising at least one clubroot resistance locus with a second Brassica plant of a different genotype to produce progeny plants; obtaining a nucleic acid-containing sample from one or more of the progeny plants; and screening the samples for a sequence comprising a molecular marker allele or a haplotype of molecular marker alleles linked to clubroot resistance at the following loci: CrB8 located on chromosome N8 interval flanked by and including 12.94 cM and 16.44 cM, CrG8 located on chromosome N8 interval flanked by and including 13.94 cM and 14.07 cM, CrE8 located on chromosome N8 flanked by and including 12.87 cM and 13.98 cM, CrM8 located on chromosome N8 interval flanked by and including 13.2 cM and 13.38 cM, or CrI8 located on chromosome N8 interval flanked by and including 13.2 cM and 13.7 cM; and selecting one or more of the progeny plants comprising the screened for molecular marker allele or haplotype, thereby obtaining a Brassica plant comprising a clubroot resistance locus.

2. The method of claim 1, wherein the one or more clubroot resistance loci physical positions on chromosome 8 (Chr 8) correspond to i) position 10,656,081 to position 13,303,318 of Chr 8; ii) position 11,124,294 to position 11,338,475 of Chr 8; iii) position 10,966,500 to position 11,249,403 of Chr 8; iv) position 10,959,267 to position 11,159,261 of Chr 8; or v) position 10,986,309 to position 11,500,321 of Chr 8 of reference line DH12075.

3. The method of claim 1, wherein the method further comprises screening the sample for the presence of the molecular marker or haplotype, wherein the molecular marker or haplotype comprises one or more CrB8 resistance alleles identified in Table 1 or Table 2 herein, one or more CrG8 resistance allele identified in Table 3 herein, one or more CrE8 resistance allele identified in Table 4 or Table 5 herein, one or more CrM8 alleles identified in Table 6 or Table 7 herein, or one or more CrI8 resistance alleles identified in Table 8 herein.

4. The method of claim 3, wherein the molecular marker or haplotype comprises one or more of the following alleles: i) N101BW0-001-Q001 (SEQ ID NO:23), N101T3M-001-Q001 (SEQ ID NO:30), N101T3P-001-Q001(SEQ ID NO:33), or N101T3R-001-Q001 (SEQ ID NO:37); ii) N100C6A-001-Q001 (SEQ ID NO:44); iii) N1000T-001-Q001 (SEQ ID NO:180), N101T3T-001-Q001 (SEQ ID NO:219), or N101T3U-001-Q001 (SEQ ID NO:222); iv) N100CDD-001-Q001 (SEQ ID NO:262), N101T3X-001-Q001 (SEQ ID NO:275), N101T3Y-001-Q001 (SEQ ID NO:278), or N101T41-001-Q001 (SEQ ID NO:282); or v) N101T0T-001-Q003 (SEQ ID NO:302).

5-7. (canceled)

8. The method of claim 1 further comprising: crossing the selected one or more progeny plants with the second parent Brassica plant to produce backcross progeny plants.

9. The method of claim 8 further comprising: obtaining a nucleic acid-containing sample from one or more backcross progeny plants; screening each sample from the backcross progeny plants for a sequence comprising the screened for molecular marker allele or a haplotype; and selecting one or more backcross progeny plants comprising the screened for molecular marker allele or haplotype.

10. The method of claim 9 further comprising: crossing the selected one or more backcross progeny plants with the second parent Brassica plant to produce additional backcross progeny plants; screening a nucleic acid-containing sample from one or more additional backcross progeny plants for a sequence comprising the screened for molecular marker allele or a haplotype; and selecting one or more additional backcross progeny plants comprising the screened for molecular marker allele or haplotype.

11. The method of claim 10, further comprising repeating steps of screening and selecting additional backcross progeny plants two or more additional times to produce further backcross progeny plants that comprise the screened for molecular marker allele or haplotype and the agronomic characteristics of the second parent plant when grown in the same environmental conditions.

12. The method of claim 1, wherein screening each sample comprises the use of a first probe comprising any probe for resistance allele sequence identified in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table7, or Table 8 herein, to thereby detect the presence of a molecular marker allele linked to clubroot resistance.

13. A method for determining zygosity of a clubroot resistance allele in a Brassica plant, cell or germplasm thereof, the method comprising: isolating nucleic acid from a Brassica plant, cell or germplasm thereof; screening the nucleic acid using a first probe comprising any probe for resistance allele sequence identified in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table7, or Table 8 herein and a second probe comprising any probe for susceptibility allele sequence identified in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table7, or Table 8 herein respectively, wherein the first probe is indicative of a marker allele linked to clubroot disease resistance, and the second probe is indicative of a maker allele linked to clubroot disease susceptibility; quantifying the binding of the first and second probe to the isolated nucleic acid sequence; and, comparing the quantified binding of the first and second probe to determine zygosity of the clubroot resistance allele.

14. The method of claim 13, wherein the method comprises: amplifying the isolated nucleic acid using a first forward primer comprising a forward primer sequence identified in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table7, or Table 8 and a first reverse primer comprising a reverse primer sequence identified in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table7, or Table 8; screening the amplified nucleic acid using the first probe and the second probe; and quantifying the binding of the first and second probe to the amplified nucleic acid sequence.

15. The method of 13, wherein the method comprises isolating nucleic acid from a Brassica plant and determining that the Brassica plant, cell or germplasm thereof, is heterozygous or homozygous for the clubroot resistance allele and the method further comprises: selecting the Brassica plant (first Brassica plant) as a parent donor crossing the first Brassica plant with a second Brassica plant to thereby produce a population of progeny plants comprising the clubroot resistance allele.

16. The method of claim 15, wherein the method comprises selecting a Brassica plant and crossing the selected Brassica plant with a second Brassica plant to thereby produce a population of progeny plants comprising the clubroot resistance allele.

17. The method of claim 1, wherein screening the sample for a sequence comprising a molecular marker allele or a haplotype of molecular marker alleles linked to clubroot resistance comprises nucleic acid sequencing, amplification, or both amplification and nucleic acid sequencing.

18. A method for obtaining a Brassica plant, cell, or germplasm thereof comprising a clubroot disease resistance locus, the method comprising: providing a population of Brassica plants, cells, or germplasm thereof; obtaining a nucleic acid containing sample from members of the population; screening the samples for a sequence comprising a molecular marker allele or a haplotype of molecular marker alleles linked to clubroot resistance at the following loci: CrB8 located on chromosome N8 interval flanked by and including 12.94 cM and 16.44 cM , CrG8 located on chromosome N8 interval flanked by and including 13.94 cM and 14.07 cM, CrE8 located on chromosome N8 flanked by and including 12.87 cM and 13.98 cM, CrM8 located on chromosome N8 interval flanked by and including 13.2 cM and

13. 38 cM, or CrI8 located on chromosome N8 interval flanked by and including 13.2 cM and 13.7 cM; selecting one or more of the Brassica plants, cells, or germplasm thereof comprising the screened for marker allele or haplotype; and testing the selected Brassica plants, cells, or germplasm thereof for resistance to clubroot disease.

19. The method of claim 18, wherein the one or more clubroot resistance loci physical positions on chromosome 8 (Chr 8) correspond to vi) position 10,656,081 to position 13,303,318 of Chr 8; vii) position 11,124,294 to position 11,338,475 of Chr 8; viii) position 10,966,500 to position 11,249,403 of Chr 8; ix) position 10,959,267 to position 11,159,261 of Chr 8; or x) position 10,986,309 to position 11,500,321 of Chr 8 of reference line DH12075.

20. The method of claim 18, wherein the method further comprises screening the sample for the presence of the molecular marker or haplotype, wherein the molecular marker or haplotype comprises one or more CrB8 resistance alleles identified in Table 1 or Table 2 herein, one or more CrG8 resistance allele identified in Table 3 herein, one or more CrE8 resistance allele identified in Table 4 or Table 5 herein, one or more CrM8 alleles identified in Table 6 or Table 7 herein, or one or more CrI8 resistance alleles identified in Table 8 herein.

21. The method of claim 18, wherein the molecular marker or haplotype comprises one or more of the following alleles: i) N101BW0-001-Q001 (SEQ ID NO:23), N101T3M-001-Q001 (SEQ ID NO:30), N101T3P-001-Q001(SEQ ID NO:33), or N101T3R-001-Q001 (SEQ ID NO:37); ii) N100C6A-001-Q001 (SEQ ID NO:44); iii) N100CJT-001-Q001 (SEQ ID NO:180), N101T3T-001-Q001 (SEQ ID NO:219), or N101T3U-001-Q001 (SEQ ID NO:222); iv) N100CDD-001-Q001 (SEQ ID NO:262), N101T3X-001-Q001 (SEQ ID NO:275), N101T3Y-001-Q001 (SEQ ID NO:278), or N101T41-001-Q001 (SEQ ID NO:282); or v) N101T0T-001-Q003 (SEQ ID NO:302).

Description

DETAILED DESCRIPTION

[0016] Terms used in the claims and specification are defined as set forth below unless otherwise specified. It must be noted that, as used in the specification and the appended claims, the singular forms a, an and the include plural referents unless the context clearly dictates otherwise.

Terms and Definitions

[0017] 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.

[0018] An amplicon is amplified nucleic acid, e.g., a nucleic acid that is produced by amplifying a template nucleic acid by any available amplification method (e.g., polymerase chine reaction (PCR), ligase chain reaction (LCR), transcription, or the like).

[0019] Backcrossing refers to the process whereby hybrid progeny plants are repeatedly crossed back to one of the parents. In a backcrossing scheme, the donor parent refers to the parental plant with the desired gene or locus to be introgressed. The recipient parent (used one or more times) or recurrent parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. Backcrossing has been widely used to introduce new traits into plants. See e.g., Jensen, N., Ed. Plant Breeding Methodology, John Wiley & Sons, Inc., 1988. In a typical backcross protocol, the original variety of interest (recurrent parent) is crossed to a second variety (non-recurrent parent) that carries a gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent, and the process is repeated until a plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent plant are recovered in the converted plant, in addition to the transferred gene from the nonrecurrent parent.

[0020] Brassica refers to any one of Brassica napus (AACC, 2n=38), Brassica juncea (AABB, 2n=36), Brassica carinata (BBCC, 2n=34), Brassica rapa (syn. B. campestris) (AA, 2n=20), Brassica oleracea (CC, 2n=18) or Brassica nigra (BB, 2n=16).

[0021] The term cross (or crossed) refers to the fusion of gametes via pollination to produce progeny (e.g., cells, seeds, and plants). This term encompasses both sexual crosses (i.e., the pollination of one plant by another) and selfing (i.e., self-pollination, for example, using pollen and ovule from the same plant).

[0022] The term 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.

[0023] The term gene (or genetic element) may refer to a heritable genomic DNA sequence with functional significance. A gene includes a nucleic acid fragment that expresses a functional molecule such as, but not limited to, a specific protein, including regulatory sequences preceding (5 non-coding sequences) and following (3 non-coding sequences) the coding sequence, as well as intervening intron sequences. The term gene may also be used to refer to, for example and without limitation, a cDNA and/or an mRNA encoded by a heritable genomic DNA sequence.

[0024] The term genome as it applies to a prokaryotic and eukaryotic cell or organism cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondria, or plastid) of the cell.

[0025] A genomic sequence or genomic region is a segment of a chromosome in the genome of a cell that is present on either side of the target site or, alternatively, also comprises the target site or a portion thereof. An endogenous genomic sequence refers to genomic sequence within a plant cell.

[0026] As used herein, gene includes a nucleic acid fragment or sequence that expresses a functional molecule such as, but not limited to, a specific protein coding sequence and regulatory elements, such as those preceding (5 non-coding sequences) and following (3 non-coding sequences) the coding sequence.

[0027] A genomic locus as used herein refers to the genetic or physical location on a chromosome of a gene.

[0028] The term genotype refers to the physical components, i.e., the actual nucleic acid sequence at one or more loci in an individual plant.

[0029] The term germplasm refers to genetic material of or from an individual plant or group of plants (e.g., a plant line, variety, and family), or 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. In general, germplasm provides genetic material with a specific molecular makeup that is the basis for hereditary qualities of the plant. As used herein, 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). Thus, germplasm is used herein synonymously with genetic material and may be used to refer to seed (or other plant material) from which a plant may be propagated. A germplasm bank may refer to an organized collection of different seed or other genetic material (wherein each genotype is uniquely identified) from which a known cultivar may be cultivated, and from which a new cultivar may be generated. In embodiments, a germplasm utilized in a method or plant as described herein is from a canola line or variety. In particular examples, a germplasm is seed of the canola line or variety. In particular examples, a germplasm is a nucleic acid sample from the Brassica line or variety.

[0030] A haplotype is the genotype of an individual at a plurality of genetic loci, i.e. a combination of alleles. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome segment.

[0031] The terms increased or improved in connection with clubroot resistance is used herein to refer to plants having increased growth, productivity, and/or reduction in root size or number of root nodules, relative a plant that is susceptible (lacking resistance) to clubroot disease, when grown in a field comprising Plasmodiophora brassicae.

[0032] The term introgression refers to the transmission of an allele at a genetic locus into a genetic background. For example, introgression of a specific allele can involve a sexual cross between two parents of the same species, where at least one of the parents has the specific allele in its genome, to thereby transfer the allele to at least one progeny. 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 progeny's genetic background. In some embodiments, introgression of a specific allele 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 in its genome. Introgression may involve transmission of a specific allele that may be, for example, a selected allele form of a marker allele, a QTL, and/or a transgene.

[0033] As used herein an isolated biological component (such as a nucleic acid or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs (i.e., other chromosomal and extra-chromosomal DNA and RNA, and proteins), while effecting a chemical or functional change in the component. For example and without limitation, a nucleic acid may be isolated from a chromosome by breaking chemical bonds connecting the nucleic acid to the remaining DNA in the chromosome and/or the other material previously associated with the nucleic acid in its cellular milieu (e.g., the nucleus). Nucleic acid molecules and proteins that have been isolated include nucleic acid molecules and proteins that are enriched or purified . The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically-synthesized nucleic acid molecules, proteins, and peptides.

[0034] Marker-assisted selection (MAS) is a process by which phenotypes are selected based on marker genotypes. Marker assisted selection can include the use of genetic markers to identify plants for inclusion in and/or removal from a breeding program or planting. A molecular marker allele that demonstrates linkage disequilibrium with a desired phenotypic trait (e.g., a QTL) provides a useful tool for the selection of the desired trait in a plant population. Components for implementing a MAS approach include the creation of a dense (information rich) genetic map of molecular markers in the plant germplasm; the detection of at least one QTL based on statistical associations between marker and phenotypic variability; the definition of a set of particular useful marker alleles based on the results of the QTL analysis; and the use and/or extrapolation of this information to the current set of breeding germplasm to enable marker-based selection decisions to be made.

[0035] The closer a particular marker is to a gene that encodes a polypeptide that contributes to a particular phenotype (whether measured in terms of genetic or physical distance), the more tightly-linked is the particular marker to the phenotype. In view of the foregoing, it will be appreciated that the closer (whether measured in terms of genetic or physical distance) that a marker is linked to a particular gene, the more likely the marker is to segregate with that gene (e.g., a clubroot disease resistance marker disclosed herein) and its associated phenotype (e.g., clubroot disease resistance disclosed herein). Thus, the tightly linked genetic markers for clubroot resistance disclosed herein can be used in MAS programs to identity Brassica varieties that have or can generate progeny that have increased clubroot resistance (relative to parental varieties and/or otherwise isogenic plants lacking that clubroot disease resistance marker), to identify individual plants comprising this clubroot disease resistance trait, and to breed this trait into other Brassica varieties to improve their clubroot disease resistance. Marker-assisted selection is discussed in more detail in a subsection hereinbelow.

[0036] 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. Thus, a set of markers linked to clubroot resistance may be used to identify a Brassica plant comprising one the clubroot disease resistance loci disclosed herein. Data corresponding to a marker set (or data derived from the use of such markers) may be stored in an electronic medium. While each marker in a marker set is useful in the identification of individuals comprising a trait of interest, subsets of markers in a set (i.e., some but not necessarily all of the markers in a marker set) can be used to effectively identify individuals comprising the trait of interest disclosed herein, i.e., one of the clubroot disease resistance loci disclosed herein.

[0037] A modified gene is a gene that has been mutated or altered through human intervention. Such a modified gene has a sequence that differs from the sequence of the corresponding non-modified gene by at least one nucleotide addition, deletion, or substitution. A modified plant is a plant comprising a modified gene or deletion.

[0038] As used herein the term native gene refers to a gene as it is found in its natural endogenous location operably linked to its own regulatory sequences, which have not been altered by human intervention. In the context of this disclosure, a modified gene is not a native gene.

[0039] As used herein, a nucleic acid molecule is a polymeric form of nucleotides, which can include both sense and anti-sense strands of RNA, cDNA, genomic DNA, recombinant and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide, or a modified form of either type of nucleotide. As used herein nucleic acid molecule is synonymous with the terms nucleic acid, nucleotide sequence, nucleic acid sequence, and polynucleotide. The term includes single- and double-stranded forms of DNA or RNA. A nucleic acid molecule can refer to either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., peptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). The term nucleic acid molecule also includes any topological conformation, including single-stranded, double stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations. An endogenous nucleic acid sequence refers to a nucleic acid sequence within a plant cell, (e.g. an endogenous allele of a native gene present within the genome of a Brassica plant cell).

[0040] The term single-nucleotide polymorphism (SNP) refers to a DNA sequence variation occurring when a single nucleotide in the genome (or other shared sequence) differs between members of a species or paired chromosomes in an individual. In some examples, markers linked to a clubroot disease resistance locus disclosed herein are SNP markers. Recent high-throughput genotyping technologies such as GoldenGate? and INFINIUM? assays (Illumina, San Diego, Calif.) may be used in accurate and quick genotyping methods by multiplexing SNPs from 384-plex to >100,000-plex assays per sample.

[0041] As used herein, phenotype means the detectable characteristics (e.g. clubroot disease resistance) of a cell or organism which can be influenced by genotype.

[0042] As used herein, the term plant material refers to any processed or unprocessed material derived, in whole or in part, from a plant. For example, and without limitation, a plant material may be a plant part, a seed, a fruit, a leaf, a root, a plant tissue, a plant tissue culture, a plant explant, or a plant cell.

[0043] As used herein, the term plant may refer to a whole plant, a cell or tissue culture derived from a plant, and/or any part of any of the foregoing. Thus, the term plant encompasses, for example and without limitation, whole plants; plant components and/or organs (e.g., leaves, stems, and roots); plant tissue; seed; and a plant cell. A plant cell may be, for example and without limitation, a cell in and/or of a plant, a cell isolated from a plant, and a cell obtained through culturing of a cell isolated from a plant. Thus, the term Brassica plant may refer to, for example and without limitation, a whole Brassica plant; multiple Brassica plants; Brassica plant cell(s); Brassica plant protoplast; Brassica tissue culture (e.g., from which a Brassica plant can be regenerated); Brassica plant callus; Brassica plant parts (e.g., seed, flower, cotyledon, leaf, stem, bud, root, and root tip); and Brassica plant cells that are intact in a Brassica plant or in a part of a Brassica plant.

[0044] As used herein, a plant or Brassica 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.

[0045] Trait or phenotype: The terms trait and phenotype are used interchangeably herein. For the purposes of the present disclosure, traits of particular interest are the clubroot disease resistance traits associated with each of the clubroot disease resistance loci disclosed herein.

[0046] A variety or cultivar is a plant line that can be used for commercial production and which is distinct 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.

[0047] Detection of Disclosed Markers. Each of the markers for the CrB8, CrG8, CrE8, CrM8, and CrI8 loci disclosed herein can be detected by any suitable method for detecting genetic polymorphisms. Suitable methods of detection include nucleotide amplification and/or sequencing of the genetic material, e.g., nucleic acid or genomic DNA sequencing that reveals the presence for a disease resistance marker allele disclosed herein for the CrB8, CrG8, CrE8, CrM8, and CrI8 loci. See Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8 (Tables 1-8) disclosing clubroot disease resistance markers alleles for each of the loci disclosed herein.

[0048] The clubroot disease resistance marker alleles can be identified and distinguished from susceptible allele using allele-specific amplification and PCR-based amplification assays such as TaqMan, rhAmp-SNP, KASPar, and molecular beacons. Such an assay can include the use of one or more probes that detect the marker allele in (i) nucleic acid that is isolated from a plant or (ii) an amplicon that is selectively amplified by amplification of nucleic acid isolated from a plant. Optionally, such an assay can further include an additional set of primers and/or one or more probes that detect the presence of a clubroot susceptible (e.g., wildtype) allele and thereby determine the zygosity (or even the absence) of clubroot resistance loci disclosed herein.

[0049] Additional methods for genotyping and detecting a resistant marker allele for the CrB8, CrG8, CrE8, CrM8, and CrI8 loci disclosed herein (or a linked marker) include but are not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, minisequencing and coded spheres. Such methods are reviewed in publications including Gut, 2001, Hum. Mutat. 17:475; Shi, 2001, Clin. Chem. 47:164; Kwok, 2000, Pharmacogenomics 1:95; Bhattramakki and Rafalski, Discovery and application of single nucleotide polymorphism markers in plants, in PLANT GENOTYPING: THE DNA FINGERPRINTING OF PLANTS (CABI Publishing, Wallingford 2001). A wide range of commercially available technologies utilize these and other methods to interrogate the allele disclosed herein (or a linked marker), including Masscode? (Qiagen, Germantown, Md.), Invader? (Hologic, Madison, Wis.), SnapShot? (Applied Biosystems, Foster City, Calif.), Taqman? (Applied Biosystems, Foster City, Calif.) and Infinium Bead Chip? and GoldenGate? allele-specific extension PCR-based assay (Illumina, San Diego, Calif).

[0050] In particular example, detecting a disclosed maker can include nucleic acid sequencing, nucleic acid amplification, or the combined amplification and nucleic acid sequencing of the marker allele and 5 bp or more, 10 bp or more, 15 bp or more, 20 bp or more, 30 bp or more, 40 bp or more, 50 bp or more, 60 bp or more, 70 bp or more, 80 bp or more, 90 bp or more, 100 bp or more, 110 bp or more, 120 bp or more, 130 bp or more, 140 bp or more, 150 bp or more, 175 bp or more, 200 bp or more, 250 bp or more, 300 bp or more, 350 bp or more, 400 bp or more, 450 bp or more, 500 bp or more, 550 bp or more, or 600 bp or more of flanking sequence that are (i) upstream of (i.e., located 5 to) the relevant marker allele and/or (ii) downstream of (i.e., located 3 to) the relevant marker allele. Thus, in particular examples, the disclosed marker can be detected by amplifying nucleic acid (e.g., genomic DNA) sequence to produce an amplicon comprising one or more of the marker allele sequences identified in Tables 1-8 herein. Primers suitable for amplification of each marker are disclosed Tables 1-8. Additionally, the markers disclosed herein can be detected by nucleotide sequencing of nucleic acids such as genomic DNA (e.g., by first amplifying genomic sequence and sequencing the resulting amplicon) comprising a resistance marker allele sequence identified in Tables 1-8 for each of the disclosed CrB8, CrG8, CrE8, CrM8, and CrI8 loci, respectively.

[0051] Other methods of detecting the marker allele for the CrB8, CrG8, CrE8, CrM8, and CrI8 loci disclosed herein include single base extension (SBE) methods, which involve the extension of a nucleotide primer that is adjacent to a polymorphism to incorporate a detectable nucleotide residue upon extension of the primer through the polymorphism, e.g., extension through the marker allele disclosed herein.

[0052] Methods of detecting the marker allele for the CrB8, CrG8, CrE8, CrM8, and CrI8 loci disclosed herein also include LCR; and transcription-based amplification methods (e.g., SNP detection, SSR detection, RFLP analysis, and others). Useful techniques include hybridization of a probe nucleic acid to a nucleic acid corresponding to a marker allele disclosed herein, or a linked marker (e.g., an amplified nucleic acid produced using a genomic canola DNA molecule as a template). Hybridization formats including, for example and without limitation, solution phase; solid phase; mixed phase; and in situ hybridization assays may be useful for allele detection in particular embodiments. An extensive guide to hybridization of nucleic acids is discussed in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes (Elsevier, N.Y. 1993).

[0053] Many detection methods (including amplification-based and sequencing-based methods) may be readily adapted to high throughput analysis in some examples, for example, by using available high throughput sequencing methods, such as sequencing by hybridization.

[0054] Detecting each of the CrB8, CrG8, CrE8, CrM8, and CrI8 loci (or marker allele therefor) disclosed herein can be done using nucleotide sequencing products, amplicons, or probes comprising detectable labels. Detectable labels suitable for use include any composition that can be detected by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Thus, 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, luminescent or phosphorescent indicators, and colorimetric labels. Other labels include ligands that bind to antibodies or specific binding targets labeled with fluorophores, chemiluminescent agents, and enzymes. In some examples the detection techniques disclosed herein include the use of fluorescent dyes (e.g. FAM, VIC, TET, FITC, TRITC, Texas Red, etc.) with or without a quencher (BHQ1 or DABsyl).

Marker Assisted Selection

[0055] Molecular markers can be used in a variety of plant breeding applications (e.g. see Staub et al. (1996) Hortscience 31: 729-741; Tanksley (1983) Plant Molecular Biology Reporter. 1: 3-8). A molecular marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is particularly true where the phenotype is hard to assay. Since DNA marker assays are less laborious and take up less physical space than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line. Thus, marker-assisted selection (MAS) has been used to significantly increase the efficiency of plant breeding at least in part by improving the efficiency of backcrossing and gene introgression.

[0056] The closer the linkage between marker and locus, the more useful the marker, as recombination is less likely to occur between the marker and the genomic feature that causes the trait, which can result in false positives. Having flanking markers on both sides of a locus decreases the chances that false positive selection will occur as a double recombination event would be needed. Generally, it is most preferred to have a marker within or at the genomic locus (e.g., within the gene or at the mutation that causes the phenotype) itself, so that recombination cannot occur between the marker and the causal gene or mutation. In some embodiments, the methods disclosed herein produce a marker in a disease resistance gene, wherein the gene was identified by inferring genomic location from clustering of conserved domains or a clustering analysis.

[0057] When a gene is introgressed by MAS, it is not only the gene that is introduced but also the flanking regions (Gepts (2002). Crop Sci; 42: 1780-1790). This is referred to as linkage drag. In the case where the donor plant is highly unrelated to the recipient plant, these flanking regions carry additional genes that may code for agronomically undesirable traits. This linkage drag may also result in reduced yield or other negative agronomic characteristics even after multiple cycles of backcrossing into the elite line. This is also sometimes referred to as yield drag. The size of the flanking region can be decreased by additional backcrossing, although this is not always successful, as breeders do not have control over the size of the region or the recombination breakpoints (Young et al. (1998) Genetics 120:579-585). In classical breeding it is usually only by chance that recombinations are selected that contribute to a reduction in the size of the donor segment (Tanksley et al. (1989). Biotechnology 7: 257-264). Even after 20 backcrosses in backcrosses of this type, one may expect to find a sizeable piece of the donor chromosome still linked to the gene being selected. With markers however, it is possible to select those rare individuals that have experienced recombination near the gene of interest. In 150 backcross plants, there is a 95% chance that at least one plant will have experienced a crossover within 1 cM of the gene, based on a single meiosis map distance. Markers will allow unequivocal identification of those individuals. With one additional backcross of 300 plants, there would be a 95% chance of a crossover within 1 cM single meiosis map distance of the other side of the gene, generating a segment around the target gene of less than 2 cM based on a single meiosis map distance. This can be accomplished in two generations with markers, while it would have required on average 100 generations without markers (See Tanksley et al., supra). When the exact location of a gene is known, flanking markers surrounding the gene can be utilized to select for recombinations in different population sizes. For example, in smaller population sizes, recombinations may be expected further away from the gene, so more distal flanking markers would be required to detect the recombination.

[0058] Important components to the implementation of MAS are: (i) defining the population within which the marker-trait association will be determined, which can be a segregating population, or a random or structured population; (ii) monitoring the segregation or association of polymorphic markers relative to the trait, and determining linkage or association using statistical methods; (iii) defining a set of desirable markers based on the results of the statistical analysis, and (iv) the use and/or extrapolation of this information to the current set of breeding germplasm to enable marker-based selection decisions to be made. The markers described in this disclosure, as well as other marker types such as SSRs and FLPs, can be used in marker assisted selection protocols.

[0059] SSRs can be defined as relatively short runs of tandemly repeated DNA with lengths of 6 bp or less (Tautz (1989) Nucleic Acid Research 17: 6463-6471; Wang et al. (1994) Theoretical and Applied Genetics, 88:1-6) Polymorphisms arise due to variation in the number of repeat units, probably caused by slippage during DNA replication (Levinson and Gutman (1987) Mol Biol Evol 4: 203-221). The variation in repeat length may be detected by designing PCR primers to the conserved non-repetitive flanking regions (Weber and May (1989) Am J Hum Genet. 44:388-396). SSRs are highly suited to mapping and MAS as they are multi-allelic, codominant, reproducible and amenable to high throughput automation (Rafalski et al. (1996) Generating and using DNA markers in plants. In: Non-mammalian genomic analysis: a practical guide. Academic press. pp 75-135).

[0060] Various types of SSR markers can be generated, and SSR profiles can be obtained by gel electrophoresis of the amplification products. Scoring of marker genotype is based on the size of the amplified fragment.

[0061] Various types of FLP markers can also be generated. Most commonly, amplification primers are used to generate fragment length polymorphisms. Such FLP markers are in many ways similar to SSR markers, except that the region amplified by the primers is not typically a highly repetitive region. Still, the amplified region, or amplicon, will have sufficient variability among germplasm, often due to insertions or deletions, such that the fragments generated by the amplification primers can be distinguished among polymorphic individuals, and such indels are known to occur frequently in maize (Bhattramakki et al. (2002). Plant Mol Biol 48, 539-547; Rafalski (2002b), supra).

[0062] SNP markers detect single base pair nucleotide substitutions. Of all the molecular marker types, SNPs are the most abundant, thus having the potential to provide the highest genetic map resolution (Bhattramakki et al. 2002 Plant Molecular Biology 48:539-547). SNPs can be assayed at an even higher level of throughput than SSRs, in a so-called ultra-high-throughput fashion, as SNPs do not require large amounts of DNA and automation of the assay may be straight-forward. SNPs also have the promise of being relatively low-cost systems. These three factors together make SNPs highly attractive for use in MAS. Several methods are available for SNP genotyping, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, minisequencing, and coded spheres. Such methods have been reviewed in: Gut (2001) Hum Mutat 17 pp. 475-492; Shi (2001) Clin Chem 47, pp. 164-172; Kwok (2000) Pharmacogenomics 1, pp. 95-100; and Bhattramakki and Rafalski (2001) Discovery and application of single nucleotide polymorphism markers in plants. In: R. J. Henry, Ed, Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing, Wallingford. A wide range of commercially available technologies utilize these and other methods to interrogate SNPs including Masscode? (Qiagen), INVADER?. (Third Wave Technologies) and Invader PLUS?, SNAPSHOT?. (Applied Biosystems), TAQMAN?. (Applied Biosystems) and BEADARRAYS?. (Illumina).

[0063] A number of SNPs together within a sequence, or across linked sequences, can be used to describe a haplotype for any particular genotype (Ching et al. (2002), BMC Genet. 3:19 pp Gupta et al. 2001, Rafalski (2002b), Plant Science 162:329-333). Haplotypes can be more informative than single SNPs and can be more descriptive of any particular genotype. For example, a single SNP may be allele T for a specific line or variety with disease resistance, but the allele T might also occur in the breeding population being utilized for recurrent parents. In this case, a haplotype, e.g. a combination of alleles at linked SNP markers, may be more informative. Once a unique haplotype has been assigned to a donor chromosomal region, that haplotype can be used in that population or any subset thereof to determine whether an individual has a particular gene. See, for example, WO2003054229. Using automated high throughput marker detection platforms makes this process highly efficient and effective.

[0064] Many of the markers presented herein can readily be used as single nucleotide polymorphic (SNP) markers to select for clubroot resistance. Using PCR, the primers are used to amplify DNA segments from individuals (preferably inbred) that represent the diversity in the population of interest. The PCR products are sequenced directly in one or both directions. The resulting sequences are aligned and polymorphisms are identified. The polymorphisms are not limited to single nucleotide polymorphisms (SNPs), but also include indels, CAPS, SSRs, and VNTRs (variable number of tandem repeats). Specifically, with respect to the fine map information described herein, one can readily use the information provided herein to obtain additional polymorphic SNPs (and other markers) within the region amplified by the primers disclosed herein. Markers within the described map region can be hybridized to BACs or other genomic libraries, or electronically aligned with genome sequences, to find new sequences in the same approximate location as the described markers.

[0065] In addition to SSR's, FLPs and SNPs, as described above, other types of molecular markers are also widely used, including but not limited to expressed sequence tags (ESTs), SSR markers derived from EST sequences, randomly amplified polymorphic DNA (RAPD), and other nucleic acid based markers.

[0066] Isozyme profiles and linked morphological characteristics can, in some cases, also be indirectly used as markers. Even though they do not directly detect DNA differences, they are often influenced by specific genetic differences. However, markers that detect DNA variation are far more numerous and polymorphic than isozyme or morphological markers (Tanksley (1983) Plant Molecular Biology Reporter 1:3-8).

[0067] Sequence alignments or contigs may also be used to find sequences upstream or downstream of the specific markers listed herein. These new sequences, close to the markers described herein, are then used to discover and develop functionally equivalent markers. For example, different physical and/or genetic maps are aligned to locate equivalent markers not described within this disclosure but that are within similar regions. These maps may be within the species, or even across other species that have been genetically or physically aligned.

[0068] In general, MAS uses polymorphic markers that have been identified as having a significant likelihood of co-segregation with a trait such as the clubroot disease resistance traits disclosed herein. Such markers are presumed to map near a gene or genes that give the plant its disease resistant phenotype, and are considered indicators for the desired trait, or markers. Plants are tested for the presence of a desired allele in the marker, and plants containing a desired genotype at one or more loci are expected to transfer the desired genotype, along with a desired phenotype, to their progeny. Thus, plants with clubroot disease resistance may be selected for by detecting one or more marker alleles, and in addition, progeny plants derived from those plants can also be selected. Hence, a plant containing a desired genotype in a given chromosomal region (i.e. a genotype associated with disease resistance) is obtained and then crossed to another plant. The progeny of such a cross would then be evaluated genotypically using one or more markers and the progeny plants with the same genotype in a given chromosomal region would then be selected as having disease resistance.

[0069] The markers disclosed herein can be used alone or in combination (i.e. as haplotype) to select for a favorable clubroot resistance locus. For example, each SNP having the resistance allele disclosed in Table 1 (e.g., N101BW0-001-Q001 having the A allele at position 10 of SEQ ID NO:1) can be used alone or in combination with another SNP resistance allele (e.g., the N101BW0-001-Q001 having T allele and N101BW2-001-Q001 having the T allele at position 12 of SEQ ID NO:5), or a combination thereof.

[0070] The skilled artisan would expect that there might be additional polymorphic sites at marker loci in and around a chromosome marker identified by the methods disclosed herein, wherein one or more polymorphic sites is in linkage disequilibrium (LD) with an allele at one or more of the polymorphic sites in the haplotype and thus could be used in a marker assisted selection program to introgress a gene allele or genomic fragment of interest. Two particular alleles at different polymorphic sites are said to be in LD if the presence of the allele at one of the sites tends to predict the presence of the allele at the other site on the same chromosome (Stevens, Mol. Diag. 4:309-17 (1999)). The marker loci can be located within 5 cM, 2 cM, or 1 cM (on a single meiosis based genetic map) of the disease resistance trait QTL.

[0071] The skilled artisan would understand that allelic frequency (and hence, haplotype frequency) can differ from one germplasm pool to another. Germplasm pools vary due to maturity differences, heterotic groupings, geographical distribution, etc. As a result, SNPs and other polymorphisms may not be informative in some germplasm pools.

[0072] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. For instance, while the particular examples below may illustrate the methods and embodiments described herein using a specific plant, the principles in these examples may be applied to any plant. Therefore, it will be appreciated that the scope of this invention is encompassed by the embodiments of the inventions recited herein and in the specification rather than the specific examples that are exemplified below. All cited patents and publications referred to in this application are herein incorporated by reference in their entirety, for all purposes, to the same extent as if each were individually and specifically incorporated by reference.

EXAMPLES

[0073] The following are examples of specific aspects of the invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the invention in any way.

[0074] Example 1: Screening for Disease Resistance. Corteva Agriscience conducted a large, nearly decade-long research program to identify new, major genetic sources of disease resistance in Brassica. This effort included large-scale genetic screens of Brassica napus (winter oilseed rape and canola), Brassica napus vegetable form (rutabaga) and Brassica rapa (Chinese cabbage and stubble turnip) species which share common genomes. Extensive inter-specific pre-breeding was carried out to introgress resistance gene sources, eliminate linkage drag, characterize their efficacy in different genetic backgrounds, and locate their genomic positions by linkage mapping. One product of this effort was the identification of the genomic hot spots and proprietary markers for clubroot resistance disclosed in the following Examples.

[0075] Example 2: Clubroot resistance locus CrB8. Major clubroot resistance locus CrB8 was identified and its genetic position was located to the interval flanked by and including 12.94 cM and 16.44 cM on chromosome N8. One source of this resistance locus has been identified in SW Rebus spring turnip rape from Sweden (see e.g., Tanhuanpaa et al., 2016, Genome 59(1): 11-21). The physical position of CrB8 was mapped using proprietary genomic maps to the locus corresponding to nucleotide position 10,656,081 to position 13,303,318 of chromosome N8 of a non-proprietary Brassica napus reference genome. Gene markers were identified within the chromosomal interval and then converted to TaqMan? (Thermo Fisher, Waltham, MA) assays. CrB8 marker name (NAME), physical position (POS), its resistance allele (RES) and susceptible allele (SUS), SEQ ID NO, and corresponding sequences for assay primers and probes are described in Table 1. These assays were tested on a canola diversity panel comprised of approximately 350 elite lines and hybrids representing the genetic diversity of the proprietary germplasm. Assays were also tested on a canola donor panel comprised of clubroot resistant donor lines. The purpose of both canola panel screenings was to confirm donor specificity of the markers. TaqMan? markers were also tested on two proprietary DH mapping population to confirm marker-trait association. Finally, the TaqMan? markers were tested on two F2 mapping populations to validate the markers' technical performance. In Table 1 and in Tables 2-8 herein, the single nucleotide polymorphism SNP for each resistance and susceptibility allele sequence is indicated by bold and underlined text.

TABLE-US-00001 TABLE1 SEQ POS ID NAME (bp) RES SUS NO: SEQUENCE FUNCTION N101BW 10910949 T G 1 TCTCTCTACAGTTTTGG FAMProbeforRES 0-001- Q001 2 TCTCTACCGTTTTGGTG VICProbeforSUS 3 CAATTTCATTATCGTATCTGCAAATT ForwardPrimer 4 TATGCGGCATTGGTTTCTTG ReversePrimer N101BW 11314585 A T 5 ATGGTGTTACTCCGCCT FAMProbeforRES 2-001- Q001 6 ATGGTGTTACACCGCC VICProbeforSUS 7 AGTGGAAGAGTTCCCTGATGAG ForwardPrimer 8 TGGACCACTATAAACGAGGCTAA ReversePrimer N101BW 11314585 A T 5 ATGGTGTTACTCCGCCT FAMProbeforRES 2-001- Q002 6 ATGGTGTTACACCGCC VICProbeforSUS 7 AGTGGAAGAGTTCCCTGATGAG ForwardPrimer 9 ACGAGGCTAATATATTCACTATTGGAG ReversePrimer N101BW 11314585 A T 5 ATGGTGTTACTCCGCCT FAMProbeforRES 2-001- Q003 6 ATGGTGTTACACCGCC VICProbeforSUS 10 ACCTACGATATATGGTTCAGTGGAA ForwardPrimer 8 TGGACCACTATAAACGAGGCTAA ReversePrimer N101BW 11314585 A T 5 ATGGTGTTACTCCGCCT FAMProbeforRES 2-001- Q004 6 ATGGTGTTACACCGCC VICProbeforSUS 10 ACCTACGATATATGGTTCAGTGGAA ForwardPrimer 9 ACGAGGCTAATATATTCACTATTGGAG ReversePrimer N101BW 11316993 A G 11 CACCACTTTGTTAAAA FAMProbeforRES 3-001- Q001 12 CACCACTTTGTCAAAA VICProbeforSUS 13 GGTGGTTTTGCCCTTGTAAA ForwardPrimer 14 CCAAATTCTGGTTCTTCTGACAA ReversePrimer N101BW 11505014 C T 15 CTACAGTATAAATTTCCAC FAMProbeforSUS 5-001- Q001 16 CCTACAGTATAAATCTC VICProbeforRES 17 CCTTAGAAATTTCACACAAGTTGATT ForwardPrimer 18 CAAGTTCTTTAAGGAAAGAGAGAGGTT ReversePrimer N101BW 11505014 C T 15 CTACAGTATAAATTTCCAC FAMProbeforSUS 5-001- Q002 16 CCTACAGTATAAATCTC VICProbeforRES 19 GAAGGAACCTTAGAAATTTCACACA ForwardPrimer 18 CAAGTTCTTTAAGGAAAGAGAGAGGTT ReversePrimer N101BW 11316941 G C 20 ATTCTCATCGCATCTT FAMProbeforRES A-001- Q001 21 TCTCATCGGATCTTT VICProbeforSUS 13 GGTGGTTTTGCCCTTGTAAA ForwardPrimer 14 CCAAATTCTGGTTCTTCTGACAA ReversePrimer N101BW 11319495 C A 22 CACGTTTTGTTTACATCG FAMProbeforSUS B-001- Q001 23 CACGTTTTGTTTCCA VICProbeforRES 24 AATAGGCTTATCACCTCCTTGTTTAA ForwardPrimer 25 GGCAGAAGTGGATGGGGTA ReversePrimer N101BW 11319495 C A 22 CACGTTTTGTTTACATCG FAMProbeforSUS B-001- Q002 23 CACGTTTTGTTTCCA VICProbeforRES 26 GCAACTAATAGGCTTATCACCTCCTT ForwardPrimer 25 GGCAGAAGTGGATGGGGTA ReversePrimer N101BW 11319505 C T 27 ATCGCTCCTGCAAC FAMProbeforSUS C-001- Q001 28 ATCGCTCCCGCAAC VICProbeforRES 24 AATAGGCTTATCACCTCCTTGTTTAA ForwardPrimer 25 GGCAGAAGTGGATGGGGTA ReversePrimer N101BW 11315059 C T 27 ATCGCTCCTGCAAC FAMProbeforSUS C-001- Q002 28 ATCGCTCCCGCAAC VICProbeforRES 26 GCAACTAATAGGCTTATCACCTCCTT ForwardPrimer 25 GGCAGAAGTGGATGGGGTA ReversePrimer

[0076] Each TaqMan? assay for this Example (as well as the remaining Examples 3-8 herein) was performed using 13.6 ?l of a primer probe mixture (18 ?M of each probe, 4 ?M of each primer) and 1000 ?l of master mix from ToughMix? kit (Quanta Beverly, Mass.). A liquid handler dispensed 1.3 ?l of the mix onto a 1536 well plate containing ?6 ng of dried DNA. The plate was sealed with a laser sealer and thermocycled in a Hydrocycler device (LGC Genomic Limited, Middlesex, United Kingdom) under the following conditions: 94? C. for 15 min, 40 cycles of 94? C. for 30 secs, 60? C. for 1 min. PCR products are measured using at wavelengths 485 (FAM) and 520 (VIC) by a Pherastar? plate reader (BMG Labtech, Offenburg, Germany). The values are normalized against ROX and plotted and scored on scatterplots utilizing the Kraken? software.

[0077] Marker N101BW0-001-Q001 was found to be particularly tightly linked to resistance locus CrB8 and was uniquely specific to resistant donor lines.

[0078] Additional TaqMan? markers were designed based on whole genome sequencing (WGS) data (Table 2). All markers were located within a 300 kb segment that does not include any of the markers identified in Table 1. Allele specificity was assessed using in silico WGS reads of the clubroot resistant donor and elite inbred susceptible germplasm. The selected markers can be used together as a haplotype. Donor specificity of the markers was determined using in silico WGS read data. Each marker (NAME), physical position (POS), its resistance allele (RES) and susceptible allele (SUS), SEQ ID NO, and corresponding sequences for assay primers and probes are described in Table 2.

TABLE-US-00002 TABLE2 POS SEQID NAME (bp) RES SUS NO: SEQUENCE FUNCTION N101T3 11021258 A T 29 ATCTGTACATGTGAAACA FAMProbeforSUS M-001- Q001 30 ATCTGTACATGAGAAAC VICProbeforRES 31 AACAAGTGTGATTCTCATTTCCAA ForwardPrimer 32 TGAGATGAAGACACATTCACACA ReversePrimer N101T3 11167528 A G 33 TTGTTTAAACTCTGGTTCC FAMProbeforRES P-001- Q001 34 TTGTTTAAGCTCTGGTTC VICProbeforSUS 35 GTGTCCCACCATTCTCTGCT ForwardPrimer 36 TCAATTGGTAGTTATAATGTTGTG ReversePrimer AGC N101T3 11167547 T C 37 TTCCACTTGTCTTGATG FAMProbeforRES R-001- Q001 38 TTCCACTTGTCTCGATG VICProbeforSUS 35 GTGTCCCACCATTCTCTGCT ForwardPrimer 36 TCAATTGGTAGTTATAATGTTGT ReversePrimer GAGC

[0079] The clubroot resistance markers in Tables 1 and 2 are very tightly linked to the CrB8 locus; each has an LOD score of 30 or greater. Furthermore, each of the markers was tested in two different mapping populations. Each test populations included at least 180 individuals. In both test populations, each of the marker alleles listed in Tables 1 and 2 demonstrated 100% association with the clubroot resistance and clubroot susceptibility traits.

[0080] Example 3: Clubroot resistance locus CrG8. Another major clubroot resistance locus CrG8 was identified and located to chromosomal interval flanked by and including 13.94 cM and 14.07 cM of chromosome N8. One source of this resistance locus has been identified in Gelria R European turnip (see, e.g., Hirai, M., 2006, Breeding Science 54: 223-229). The physical position of CrG8 was mapped using proprietary genomic maps to the locus corresponding to nucleotide position 11,124,294 to position 11,338,475 of chromosome N8 of a non-proprietary Brassica napus reference genome. Genetic markers located within the chromosomal interval were converted to TaqMan? assays. Each CrG8 marker (NAME), physical position (POS), its resistance allele (RES) and susceptible allele (SUS), SEQ ID NO, and corresponding sequences for assay primers and probes are described in Table 3. These assays were tested on a canola diversity panel comprised of approximately 350 elite lines and hybrids representing the genetic diversity of the proprietary germplasm and a clubroot donor panel comprised of clubroot resistant donor lines. The purpose of the panel screenings was to confirm donor specificity of the markers. The TaqMan? markers were also tested on a proprietary DH mapping population to confirm the marker-trait association and four proprietary F2 mapping populations to evaluate the markers' technical performance.

TABLE-US-00003 TABLE3 POS SEQID NAME (bp) RES SUS NO: SEQUENCE FUNCTION N100C5 11124294 T C 39 CTTATATCAATCGTGATTTC FAMProbeforSUS V-001- Q001 40 CTCTTATATCAATCATGATTTC VICProbeforRES 41 CTCGATCCATAATGTTTCTA ForwardPrimer ATCAAAAGGC 42 CCTCAGATTCCCTTATCTTGTCGAT ReversePrimer N100C6 11338475 A T 43 CCTCACAAAACAAAG FAMProbeforSUS A-001- Q001 44 TCCCTCACAATACAAAG VICProbeforRES 45 CCAAAGGATGTGACAGAGAGGTAAA ForwardPrimer 46 ACAGATGAACAAAACATGATATAACAG ReversePrimer ACTCTT N100C7 11153988 C T 47 TTTTAGTTAACATTGATTTATTAC FAMProbeforSUS R-001- Q001 48 ATTTTAGTTAACATTGATTTGTTAC VICProbeforRES 49 CTGTTGAGAAAAAATCTAACAAAATCTT ForwardPrimer ACTTAAAAATTT 50 CACATATCACTTCTATTTTTATATAA ReversePrimer TACCGAATAGAATTATAGAAT N100C7 11123059 G A 51 TCCGGAGTAAAGAGTG FAMProbeforSUS Y-001- Q001 52 CCGGAGTGAAGAGTG VICProbeforRES 53 GCCTCTTTTAGGTTTGGGTTGGA ForwardPrimer 54 CCGGCCCAGATGGGTTAAA ReversePrimer N100C8 11338868 T C 55 CTTAGTTTTGGAACGCACCA FAMProbeforSUS 4-001- Q001 56 CTTAGTTTTGGAACGCATCA VICProbeforRES 57 CCCACGGAAAAGTCTATACAACTGA ForwardPrimer 58 GTCGTCGTGGTTGTGATGATATCT ReversePrimer N100C8 11270978 A G 59 TGGCGTATAAGAAGCAATAA FAMProbeforSUS 7-001- Q001 60 TGGCGTATAAGAAACAATAA VICProbeforRES 61 TCAATACTAGGTATATACATACTTGTTT ForwardPrimer GCTAAGTGA 62 CTAGAGTGTCTACGCATTTTGAAGAGA ReversePrimer N100C8 11339261 A G 63 ACACTCAGCAAAGCA FAMProbeforSUS 6-001- Q001 64 CACACTCAACAAAGCA VICProbeforRES 65 GCAAATAACAAATCCAGACAGAACCAA ForwardPrimer 66 TGCAACCTTGTCCTATCAGTTCAAT ReversePrimer N100C7 11359435 G T 67 AAACTTGTTTATTTTTCG FAMProbeforSUS M-001- Q001 68 CAAACTTGTTTAGTTTTCG VICProbeforRES 69 ATCTTCACATTCCTGCATTGTTTTTGTT ForwardPrimer 70 ATTGTTGAAAGTTTTAGCTGTTTCAAAT ReversePrimer TAACAT N100CA 10862403 G A 71 TTCGATTTATCTCTTTTTTTT FAMProbeforSUS M-001- Q001 72 TTTCGATTTATCTCTCTTTTTT VICProbeforRES 73 AGACTGCGGTATCAGGTAAAAACAA ForwardPrimer 74 CGAACTCGAGAGCCAATCCAAATT ReversePrimer N100CA 11380539 G A 75 TTTAGTAGCCAATCATGATT FAMProbeforSUS N-001- Q001 76 TTTAGTAGCCAGTCATGATT VICProbeforRES 77 CGTTAACATTTCATTGGTTAAATTAGCG ForwardPrimer TTT 78 CATATTACGGTTTATCTTGGGTAAGAGG ReversePrimer TTAAAT N100CA 11384118 G A 79 CACCAAGAAACAAAAA FAMProbeforSUS P-001- Q001 80 CATCACCAAGAAACGAAAA VICProbeforRES 81 CGGAAGGATGATGATGAAGTGAAATAC ForwardPrimer 82 GATTCAGTTTCGCTTCATCTTCGTT ReversePrimer N100CA 11384154 G A 83 ACTGAATCAAAACAAAAG FAMProbeforSUS R-001- Q001 84 ACTGAATCAAAGCAAAAG VICProbeforRES 85 ACAGATTCTACAGGATCATCACCAAGA ForwardPrimer 86 GCTTCGATTGATCTGGATTCAAGCT ReversePrimer N100C5 11384646 T C 87 CACATTTTCAAATTATG FAMProbeforSUS P-001- Q001 88 AGTCACATTTTTAAATTATG VICProbe 89 AGTAACGCGGATTTGTGAGTCAA ForwardPrimer 90 GCCGGGCTGTCAGTACA ReversePrimer N100CA 11384688 G T 91 TTGAAGCCTGTATTTTAGT FAMProbeforSUS T-001- Q001 92 TTGAAGCCTGTAGTTTAGT VICProbeforRES 93 GCGTTTTAACTTTTAAGAGGTAGCTTGT ForwardPrimer 94 GGCCGGGCTGTCAGTAC ReversePrimer N100C5 11385653 A T 95 CAGATTTTTGGTATTGTTTT FAMProbeforSUS R-001- Q001 96 TTTCAGATTTTTGGTTTTGTTTT VICProbeforRES 97 CCCGATAATTAATAAAACCCCAATGCA ForwardPrimer A 98 CCGTCGAATTCAGTTTGGTTGATTT ReversePrimer N100C5 11386285 T C 99 CTGATGTTCGTTCTATGTC FAMProbeforSUS T-001- Q001 100 ACTGATGTTCGTTTTATGTC VICProbeforRES 101 GAATACAAAAATTCTTCAACTTGAAACT ForwardPrimer TTGGAC 102 ACTAGCAGCAAAATATCAAAATTTCAA ReversePrimer AGCA N100C8 11341835 C T 103 TCAAATAGGAGACGCATCT FAMProbeforSUS 1-001- Q001 104 CAAATAGGAGGCGCATCT VICProbeforRES 105 GAGGCATTCTCCTCTTTCACCA ForwardPrimer 106 CTGGAATCAATTACATCACAACTTTATC ReversePrimer AG N100CA 11391926 T C 107 TTTTAATTATTCAGATTATTTTT FAMProbeforSUS U-001- Q001 108 ATTTTTAATTATTCAAATTATTTTT VICProbeforRES 109 TCTTTATTAAACGGAAGAAGTATGTAAT ForwardPrimer T 110 CTGCAATTTGGTTCAGAAAATAAAACTT ReversePrimer CTAGTAA N100CA 11392121 T G 111 CGAAAACCCGAAACC FAMProbeforSUS V-001- Q001 112 CCGAAAAACCGAAACC VICProbeforRES 113 GGCTGGGCTTGTACACATGTTAATA ForwardPrimer 114 CTTAACACATTGGGCCTCAAAGG ReversePrimer N100CA 11392588 G C 115 ACCTTGTTGTACTTAGCA FAMProbeforSUS W-001- Q001 116 ATACCTTGTTGTAGTTAGCA VICProbeforRES 117 TGATAAAAAGATTTAGGATATATTACAA ForwardPrimer AACTTGACCATCA 118 CATTGTAGATGCCTAGGGTTTAAAAGTC ReversePrimer TAT N100C6 11392602 T A 119 CATATGACCAAATTTTTTT FAMProbeforSUS E-001- Q001 120 CATATGACCAAAATTTTTT VICProbeforRES 121 CAAAACTTGACCATCAATACCTTGTTGT ForwardPrimer 122 GCCATTGTAGATGCCTAGGGTTTAA ReversePrimer N100CA 10654358 G A 123 ATTTTAAAAAATTTATTATTAATTTT FAMProbeforSUS X-001- Q001 124 TTTTAAAAAATTTATTGTTAATTTT VICProbeforRES 125 TTGTTTAATAAATCAGTTTTTATGGGTT ForwardPrimer AA 126 TCAACTTAAAGATTTTCAGATTTGTAGA ReversePrimer TAATTTTTGTTA N100C6 10655493 A G 127 TTTTCAACAACTATTCTTG FAMProbforSUS 0-001- Q001 128 ATTTTTTCAACAATTATTCTTG VICProbeforRES 129 GGAGGCCACCTGGACATT ForwardPrimer 130 AAGAAATATTTTTATTATCAGATGACTA ReversePrimer TTCCGTGTTTATATACA N100CA 10471329 A G 131 ATACTGGGAAAATTT FAMProbeforSUS Y-001- Q001 132 CATATATACTGGAAAAATTT VICProbeforRES 133 ACTTACAAAATATGTATCCTGACTTTTC ForwardPrimer ATGGT 134 AGTATGAGATTGATTGGGTTTATAAATA ReversePrimer TTATATA N100C6 10473378 C G 135 CCCAAAGGATCTAAGAAA FAMProbeforSUS G-001- Q001 136 CCCAAAGGATGTAAGAAA VICProbeforRES 137 TTTATGCAATCATTGGCAACACACA ForwardPrimer 138 CCAGCCGAGAAAGACAACTTGA ReversePrimer N100C6J 10538447 T C 139 CGTCCAAATATATTGGTGGAG FAMProbeforSUS -001- Q001 140 AGACGTCCAAATATATTAGTGGAG VICProbeforRES 141 TGGAGGACCAGATTCTGTTTGG ForwardPrimer 142 TGGCGAAAAAGTCTTTATCCTTTAATTT ReversePrimer GAC

[0081] Marker N100C6A-001-Q001 was found to be particularly tightly linked to resistance locus CrG8 and was uniquely specific to resistant donor lines.

[0082] The clubroot resistance markers in Table 3 are very tightly linked to the CrG8 locus; each has an LOD score of 30 or greater. Furthermore, each of the markers was tested in two different mapping populations. Each test populations included at least 180 individuals. In both test populations, each of the marker alleles listed in Tables 1 and 2 demonstrated 100% association with the clubroot resistance and clubroot susceptibility traits.

[0083] Example 4: Clubroot resistance locus CrE8. An additional major clubroot resistance locus, CrE8 was identified and its genetic position was located to interval flanked by and including 12.87 cM and 13.98 cM on chromosome N8. The physical position of CrE8 was mapped using proprietary maps to the locus corresponding to nucleotide position 10,966,500 to 11,124,403 of chromosome N8 of a non-proprietary Brassica napus reference genome. Genetic markers linked to CrE8 were converted to TaqMan? assays. Each CrE8 marker (NAME), physical position (POS), its resistance allele (RES) and susceptible allele (SUS), SEQ ID NO, and corresponding sequences for assay primers and probes are described in Table 4. The assays and tested on a canola diversity panel comprised of approximately 350 elite lines and hybrids representing the genetic diversity of the proprietary germplasm. The purpose of panel screening was to confirm donor specificity of the markers. TaqMan? markers were also tested on two proprietary DH mapping populations to confirm marker-trait association and an F2 mapping population to validate the markers' technical performance. None of the markers overlap with publicly available 56 k array markers available from Illumina (Madison, Wisc. USA).

TABLE-US-00004 TABLE4 POS SEQ (bp) ID NAME RES SUS NO: SEQUENCE FUNCTION N100CJ0- 10966467 A T 143 ATTTGTTCTCCCTCACAATA FAMProbeforSUS 001-Q001 144 ATTTGTTCTCCCACACAATA VICProbeforRES 145 CAAGCAGTGGACTATGGTTGGTTA ForwardPrimer 146 GAGAACCTTCCTCTGTTTCAAACCT ReversePrimer N100CJI- 10970941 T C 147 TCCATCGCATTTTT FAMProbeforSUS 001-Q001 148 CTTTCCATCACATTTTT VICProbeforRES 149 GCATGCTGACGTAAACAACTACAT ForwardPrimer T 150 CGAATAACTGAGTCACGCTTCCT ReversePrimer N100CJ2- 10974310 A T 151 AAGTTACTCAGACACTCTAC FAMProbeforSUS 001-Q001 152 CAAAGTTACTCAGACTCTCTAC VICProbeforRES 153 TGGTATGTGTGGAGAGTCTGAAGT ForwardPrimer T 154 ACACGATTGTGGACGGATGAATTA ReversePrimer T N100CJ3- 10975706 G A 155 CTGATTCACCTCTCTCGAC FAMProbeforSUS 001-Q001 156 CTGATTCACCTCCCTCGAC VICProbeforRES 157 GTGTCCACATGCTCAAGAGGTT ForwardPrimer 158 GTATGCTGCAAATCGATCAGATGT ReversePrimer G N100CJ4- 11029403 T C 159 TCCACTGGCTTCTCGTTA FAMProbeforSUS 001-Q001 160 ATCCACTGGCTTTTCGTTA VICProbeforRES 161 TCATGATTTTAAACTTAACCCTGCT ForwardPrimer CCTT 162 GCATATGACTCTGTTTATCTTCCCT ReversePrimer TGT N100CJ5- 11060466 A T 163 CTAAGGGATGATAAAGGA FAMProbeforSUS 001-Q001 164 TTCTAAGGGATGATAATGGA VICProbeforRES 165 GCATCCCGCTCCCAAGAAA ForwardPrimer 166 CACCAAGAGATTGGATCAAATGTA ReversePrimer ATTATTATTATAGTTC N100CJ6- 11101333 C T 167 CAGCGTTTCATATATTTTTGGAT FAMProbeforSUS 001-Q001 168 CAGCGTTTCATATATCTTTGGAT VICProbeforRES 169 TTTTTGAGCTAATGGGCCTTCTCT ForwardPrimer 170 GGTAGGTTTCGTAGGGTAAAAGCT ReversePrimer N100CJ7- 11124993 T A 171 ACATCTCTCTCATAAAC FAMProbeforSUS 001-Q001 172 ACATCTCTCACATAAAC VICProbe 173 TGATTATTAGGGTTTTAATGTGGTG ForwardPrimer GATTGT 174 GCATCAAGGTGCCTTCTTTAACATG ReversePrimer N100CJP- 10927691 A G 175 CCCTCTGTTCGACTACA FAMProbeforSUS 002-Q001 176 ATCCCTCTGTTTGACTACA VICProbeforRES 177 ATCAGAGACTGAGTCTGCATATCC ForwardPrimer A 178 TCCTCGCATCTTCAAAACTAGTGTT ReversePrimer N100CJT- 10966500 T C 179 CTGTTTCAAACCTGAATGT FAMProbeforSUS 001-Q001 180 CTGTTTCAAACCTAAATGT VICProbeforRES 181 AAGCAGTGGACTATGGTTGGTTAA ForwardPrimer T 182 TCTCACTCAAATGGATTGTGTTCAT ReversePrimer GT N100CJV- 10973102 G T 183 AGCCGTAAACTAATTAGAG FAMProbeforSUS 002-Q001 184 CAGCCGTAAACTACTTAGAG VICProbeforRES 185 CTATTCACTTTCAATAATGGCTACG ForwardPrimer TTGC 186 CAGGCGAGAAGTATGTAAAGTCGT ReversePrimer T N100CJX- 10975316 T A 187 TGGCGGATCTCAAATT FAMProbeforSUS 002-Q001 188 CGTGGCGGATCTCATATT VICProbeforRES 189 TGTTTGTTTCTTTTGTGGGTTTTGTG ForwardPrimer A 190 TGAACCTTGATATCATCGTTGTAGA ReversePrimer CACTATAATA N100CK0- 10975456 G T 191 ATTTTGTTGTATGAGCTTT FAMProbeforSUS 002-Q001 192 ATTTTGTTGTAGGAGCTTT VICProbeforRES 193 GGTGGCTTTGAAATTTATCTTAGTA ForwardPrimer GGTCTT 194 ATTGTGAATCCCATAACGCTTAAG ReversePrimer GT N100CK2- 10975634 C T 195 AACTCTGCAAAGCTT FAMProbeforSUS 001-Q001 196 AACTCTGCGAAGCTT VICProbeforRES 197 GCTCGATGCCATCTCGTCTAG ForwardPrimer 198 CTCTTGAGCATGTGGACACTGA ReversePrimer N100CK4- 10975658 G A 199 CGGCCTGGCCCC FAMProbeforSUS 001-Q001 200 CGGCCCGGCCCC VICProbeforRES 201 GATGCCATCTCGTCTAGTAAGCTT ForwardPrimer 198 CTCTTGAGCATGTGGACACTGA ReversePrimer N100CK6- 10976300 G A 202 ACTTATTTTAAATCAAAAGTG FAMProbeforSUS 001-Q001 203 TGTACTTATTTTAAATCGAAAGTG VICProbeforRES 204 AGTTTTGGCAAATTAATTGGAGAG ForwardPrimer TAGGT 205 CGACCTTATCAATGAGAGACAAAA ReversePrimer TAATATTAGCA N100CK8- 10977693 C T 206 CCAACCAAGAAAAT FAMProbeforSUS 002-Q001 207 ATCCAACCAGGAAAAT VICProbeforRES 208 GTGTCCATCGTCATGAAGATCTCT ForwardPrimer 209 CAAGTGCCCTTTGTTGAGATTCC ReversePrimer N100CKA- 11029667 C G 210 ACGCAAAAACACTCTGATAA FAMProbeforSUS 001-Q001 211 ACGCAAAAACACTCTCATAA VICProbeforRES 212 GTTTGAAACTGAAAAAGAGTAGTA ForwardPrimer AGCACAT 213 GCAAATCACATGTAGCGTTTAAGG ReversePrimer T N100CKC- 11124709 C A 214 CGCGACTCACGCG FAMProbeforSUS 002-Q001 215 CGCGACGCACGCG VICProbeforRES 216 ACAGAGGCGGGAAGTGTTTATTT ForwardPrimer 217 TCTTCTTCTTCGTTCGTTTCGGAAA ReversePrimer

[0084] Marker N100CJT-001-Q001 was found to be particularly tightly linked to resistance locus CrE8 and was uniquely specific for resistant donors.

[0085] Additional TaqMan? markers were designed based on WGS data (Table 5). All markers are located within a 300 kb segment that does not include any of the markers identified in Table 4. Allele specificity was assessed using in silico WGS reads of the donor and elite inbred germplasm. The selected markers can be used together as a haplotype. Each marker's name (NAME), physical position (POS), its resistance allele (RES) and susceptible allele (SUS), SEQ ID NO, and corresponding sequences for assay primers and probes are described in Table 5.

TABLE-US-00005 TABLE5 SEQ POS ID NAME (bp) RES SUS NO: SEQUENCE FUNCTION N101T3T- 11290742 C T 218 CAGAACTG FAMProbe 001-Q001 ATGAGTTC forSUS 219 CAGAACTG VICProbe ATGAGTCC forRES 220 GGAAAATGC Forward AAGGAAGAG Primer CA 221 TGAATGATC Reverse TCTTTGCTG Primer TGAAA N101T3U- 11290750 A G 222 TTGCTGTGA FAMProbe 001-Q001 AATTTTTAA forRES G 223 TTGCTGTGA VICProbe AATTTTCAA forSUS G 224 CACAAGCAA Forward TTTCAAAGA Primer AGCA 225 TCTCCAATG Reverse AAAGAAAAG Primer ATTGG

[0086] The clubroot resistance markers in Tables 4 and 5 are very tightly linked to the CrE8 locus; each has an LOD score of 30 or greater. Furthermore, each of the markers was tested in two different mapping populations. Each test populations included at least 180 individuals. In both test populations, each of the marker alleles listed in Tables 4 and 5 demonstrated 100% association with the clubroot resistance and clubroot susceptibility phenotype.

[0087] Example 5: Clubroot resistance locus CrM8. One more major clubroot resistance locus CrM8 was identified and its genetic position located to the interval flanked by and including 13.2 cM and 13.38 cM on chromosome N8. One source of this resistance locus has been identified in the Brassica napus variety Mendel (see e.g., Fredua-Agyeman et al., 2016, Euphytica 211: 201-213). The physical position of CrM8 was mapped using proprietary maps to the locus corresponding to nucleotide position 10,959,267 to position 11,159,261 of chromosome N8 of a non-proprietary Brassica napus reference genome. Genetic markers located within this chromosomal interval were converted to TaqMan? assays. Each marker name (NAME), physical position (POS), its resistance allele (RES) and susceptible allele (SUS), SEQ ID NO, and corresponding sequences for assay primers and probes are described in Table 6. The assays were tested on a Brassica napus diversity panel comprised of approximately 350 elite lines and hybrids representing the genetic diversity of the proprietary germplasm and a clubroot donor panel comprising clubroot resistant donor lines. The purpose of the panel screenings was to confirm donor specificity of the markers. TaqMan? markers were also tested on two proprietary DH mapping population to confirm the marker-trait association and four F2 mapping populations to validate the markers' technical performance. None of the markers overlap with markers on publicly available 56 k array from Illumina.

TABLE-US-00006 TABLE6 POS SEQ NAME (bp) RES SUS IDNO SEQUENCE FUNCTION N100CCT 10959267 A T 226 CAAGAGAAAAGAAAGTACTAC FAMProbeforSUS -001- Q001 227 AAGAGAAAAGAAAGAACTAC VICProbeforRES 228 GGACGAACAGGACTCAAAACTCTAT ForwardPrimer A 229 GCGCTAACCCCTTTCAAATTCTTAT ReversePrimer N100CCV 11101444 G C 230 TGTATTTTCCTTTGACAGTAA FAMProbeforRES -001- Q001 231 CTGTATTTTCCTTTCACAGTAA VICProbeforSUS 232 TTGATGCTACGTATCGAATAAGAAAT ForwardPrimer GAATAGAA 233 ATCTTGGAAACCCTCTTTGGTGTT ReversePrimer N100CD4 11141347 C T 234 ACCACCAACAAATAA FAMProbeforSUS -001- Q001 235 CCACCAGCAAATAA VICProbeforRES 236 TGAGGACTGACAGAATGCACAAG ForwardPrimer 237 GAGGTAGTGTACATTTGCGACGAT ReversePrimer N100CD7 11145103 T A 238 ATCTAAGAAACTTTAATTAAAA FAMProbeforRES -001- Q001 239 AGATCTAAGAAACTTTTATTAAAA VICProbeforSUS 240 GCTTGTCAATGCCTTCCTTGTTA ForwardPrimer 241 CACATTGAGGTCCATTGATAATATTA ReversePrimer GGATGTTA N100CD8 11145490 G C 242 CTAGAGAGTATCAAACATC FAMProbeforRES -001- Q001 243 CTAGAGAGTATGAAACATC VICProbeforSUS 244 ATTTTGTTTGTTTCGTTTGAGTCTTAT ForwardPrimer CGT 245 TTGACGACTTAATGCATTCACTGAGA ReversePrimer N100CD9 11147445 A G 246 CACCACGTGTTAGTG FAMProbeforSUS -001- Q001 247 CCACCACATGTTAGTG VICProbeforRES 248 TTAACTTTTTTTTTCTTTTATTAACCA ForwardPrimer ATCGCG 249 GGCAAGTTTGGTGAGTTCTTATGGT ReversePrimer N100CD 11147528 T G 250 TTTAATAAATTTGTGGGACCC FAMProbeforRES A-001- Q001 251 AATAAAGTTGTGGGACCC VICProbeforSUS 252 CCAAACTTGCCTCTTGCAGAAG ForwardPrimer 253 TTAGAGCATCATTAACCCCACCTTTT ReversePrimer N100CDB 11147856 T C 254 CTCACAAGGTGCATACA FAMProbeforRES -001- Q001 255 ACTCACAAGGTGCACACA VICProbeforSUS 256 CTCACAAGGTGCACTGTTTCAC ForwardPrimer 257 GGCTTCCAGTCCACAATTATTCCA ReversePrimer N100CDC 11150527 C T 258 CATAGTAGTCCACATGAGTAT FAMProbeforSUS -001- Q001 259 CATAGTAGTCCACGTGAGTAT VICProbeforRES 260 ACCTTAATCAGTAGACTATAGCGCTT ForwardPrimer CT 261 GGTTGCTCAATATCGAGACTTTCTTC ReversePrimer T N100CD 11150839 G C 262 TTTTCAAAGTACCCCTAATC FAMProbeforRES D-001- Q001 263 TTTCAAAGTACGCCTAATC VICProbeforSUS 264 GTTGTGCACTAATGCATCTCACATT ForwardPrimer 265 ATGTTCATGTATTGCTCTGCTTTAGTC ReversePrimer T N100CDF 11159141 G A 266 CAGTGGATGCTATGCG FAMProbeforRES -001- Q001 267 TCAGTGGATGTTATGCG VICProbeforSUS 268 TTGTATCCACCAAATGGCATCCA ForwardPrimer 269 AATAGAGAAGTTGGGCAAGTAAAAG ReversePrimer AGATT N100CD 11159261 A G 270 CTTGACCAAACCTTATG FAMProbeforSUS G-001- Q001 271 CTTGACCAAACTTTATG VICProbeforRES 272 TTTTCATGTCAATATTCCCCCTCAAG ForwardPrimer T 273 GAGGGATGTCTTCATGGTTTCCAA ReversePrimer

[0088] Marker N100CDD-001-Q001 was found to be particularly tightly linked to resistance locus CrM8 and was uniquely specific for resistant donor lines.

[0089] Additional TaqMan? markers were designed based on WGS data (Table 7). All markers were located within a 300 kb segment that does not include any of the markers identified in Table 6. Allele specificity was assessed using in silico WGS reads of the donor and elite inbred germplasm. The selected markers can be used together as a haplotype. Each additional CrM8 marker's name (NAME), physical position (POS), its resistance allele (RES) and susceptible allele (SUS), SEQ ID NO, and corresponding sequences for assay primers and probes are described in Table 7.

TABLE-US-00007 TABLE7 POS RES SUS SEQID NAME (bp) NO SEQUENCE FUNCTION N101T3X- 11341556 G A 274 TTCGTCCTAGCAACCA FAMProbeforSUS 001-Q001 275 TTCGTCCTAGCGACCA VICProbeforRES 276 TTTTCATTCAAAAGATCAAAATCA ForwardPrimer 277 CAACGTGAAATGCAGGTGA ReversePrimer N101T3Y- 11341804 T G 278 CCTCTTTCACCATTATA FAMProbeforRES 001-Q001 279 CTCTTTCACCAGTATAT VICProbeforSUS 280 CCGGATGGAACAGTTCTTTG ForwardPrimer 281 GGAATCAATTACATCACAA ReversePrimer CTTTATCAG N101T41- 11478087 A G 282 TAGCTCCAATTGGTTTT FAMProbeforRES 001-Q001 283 TAGCTCCAGTTGGTTTT VICProbeforSUS 284 GAGGAGCACGGAACAAGATT ForwardPrimer 285 ACTTGGTCGGCCCAAACTA ReversePrimer

[0090] The clubroot resistance markers in Tables 6 and 7 are very tightly linked to the CrM8 locus; each has an LOD score of 30 or greater. Furthermore, each of the markers was tested in two different mapping populations. Each test populations included at least 180 individuals. In both test populations, each of the marker alleles listed in Tables 6 and 7 demonstrated 100% association with the clubroot resistance and clubroot susceptibility phenotype.

[0091] Example 6: Clubroot resistance locus CrI8. Yet another major clubroot resistance locus, CrI8, was identified and its genetic position was located to the interval flanked by and including 13.2 cM and 13.7 cM on chromosome N8. CrI8 was mapped using proprietary maps to the locus corresponding to nucleotide position 10,986,309 to position 11,500,321 of chromosome N8 of a non-proprietary Brassica napus reference genome. Genetic markers located within the chromosomal interval were converted to TaqMan? assays. Each marker's name (NAME), physical position (POS), its resistance allele (RES) and susceptible allele (SUS), SEQ ID NO, and corresponding sequences for assay primers and probes are described in Table 8. The assays were tested on a Brassica napus (canola/oilseed) diversity panel comprised of approximately 350 elite lines and hybrids representing the genetic diversity of the proprietary germplasm and a clubroot donor panel comprised of clubroot resistant donor lines. The purpose of the panel screenings was to confirm donor specificity of the markers. TaqMan? markers were also tested on a proprietary DH mapping population to confirm the marker-trait association and a proprietary F2 mapping population to validate the technical performance of the TaqMan? assays.

TABLE-US-00008 TABLE8 SEQ POS ID NAME (bp) RES SUS NO: SEQUENCE FUNCTION N101TO 10995424 G A 286 AATTTTTGAATATCAATTTT FAMProbeforRES M-001- Q001 287 TGAAATTTTTGAATATTAATTTT VICProbeforSUS 288 CCTAGGGTATCAATTTTTAGTTTTTTTTAC ForwardPrimer TAAATGGT 289 CTCGCAAATAATTTTCTTAAGTTTTTGTTA ReversePrimer CCAAA N101TO 10996913 C G 290 ACCATTCGCGTTTTG FAMProbeforRES N-001- Q001 291 ACCATTCGGGTTTTG VICProbeforSUS 292 TTTTTCGGGTTTGGAAATATAGGA ForwardPrimer 293 ACCCGAAAACCAAACCAAAACC ReversePrimer N101TO 10996942 C T 294 CCGATTTCGGTCTTAGTT FAMProbeforRES P-001- Q001 295 TCCGATTTCGGTTTTAGTT VICProbeforSUS 296 GGGTTTGGAAATATAGGAACCATTCG ForwardPrimer 297 ACCCGAAAACCAAACCAAAACC ReversePrimer N101TO 10996958 T C 298 AACCAAAACCAAACCGA FAMProbeforRES R-001- Q001 299 AAACCAAAACCGAACCGA VICProbeforSUS 296 GGGTTTGGAAATATAGGAACCATTCG ForwardPrimer 300 TTTAATCTAGAATCTCGTTTAGTTCTGGGC ReversePrimer N101TO 11155547 G A 301 CTTGACAAAATATAAGGTT FAMProbeforSUS T-001- Q003 302 CTTGACAAAGTATAAGGTT VICProbeforRES 303 CATGCAATCTTCCAAACTTAAAAAT ForwardPrimer 304 GTTATTCTTTATTATCTATGGTTTTATCTTT ReversePrimer TG N101TO 11191065 G A 305 CACAAACCGAACCAA FAMProbeforRES U-001- Q001 306 TTACACAAACCAAACCAA VICProbeforSUS 307 AATTGGACTCAAAATTATCTTAAATATTA ForwardPrimer GTTGGT 308 GGGTATAGGTTCGGTTTTATTTGTTCTAGA ReversePrimer N101TO 11191077 G A 309 CAAATACCGAAATAAC FAMProbeforRES V-001- Q001 310 CCAAATACCAAAATAAC VICProbeforSUS 307 AATTGGACTCAAAATTATCTTAAATATTA ForwardPrimer GTTGGT 308 GGGTATAGGTTCGGTTTTATTTGTTCTAGA ReversePrimer NP1 11368007 C A 311 GGTAACATGTATTCATC FAMProbeforSUS 312 GGTAACATGTCTTCATC VICProbeforRES 313 TTTGTAGTTGAACAAAGTTGAAGGA ForwardPrimer 314 AGGGTACGTTGGAAGGGTCT ReversePrimer NP2 11384573 A G 315 GTAACGCAGATTTGT FAMProbeforRES 316 GTAACGCGGATTTG VICProbeforSUS 317 CGGCACTAGAATACGATTCCTC ForwardPrimer 318 AAATGTGACTTAAACAAGCTACCTCTT ReversePrimer NP3 11391245 T G 319 GTTTGACGTAAAGAAA FAMProbeforRES 320 GTTTGACGGAAAGAA VICProbeforSUS 321 GGAGGAAGAGATCGGTGATG ForwardPrimer 322 TGGTAGATGAAACATCCAAGCA ReversePrimer NP4 11399507 A G 323 GATCACTCAGTTAAAT FAMProbeforRES 324 GATCACTCGGTTAAA VICProbeforSUS 325 TGGCAATTCCCCATAAATAAA ForwardPrimer 326 TGTTCATGGTTTTGAAAGTGAAA ReversePrimer NP5 11406704 A T 327 TACTAGAATGCAACCTT FAMProbeforRES 328 TACTAGTATGCAACCTT VICProbeforSUS 329 TCAGATTCCAGGATCGAGGT ForwardPrimer 330 GCTCCACTCGAAATCGTCAC ReversePrimer

[0092] Marker N101T0T-001-Q003 was found to be particularly tightly linked to resistance locus CrI8 and was uniquely specific for resistant donor lines.

[0093] The clubroot resistance markers in Table 8 are very tightly linked to the CrI8 locus; each has an LOD score of 30 or greater. Furthermore, each of the markers was tested in two different mapping populations. Each test populations included at least 180 individuals. In both test populations, each of the marker alleles listed in Table 8 demonstrated 100% association with the clubroot resistance and clubroot susceptibility phenotype.