RU2834673C2 - Snp markers and selection for low fiber content in members of genus brassica - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a method for identifying a Brassica napus plant sample containing a low fibre content, comprising obtaining a nucleic acid sample from a Brassica napus plant; sample screening for nucleic acid containing one or more marker alleles associated with low fibre content, located in chromosomal interval N13 in Brassica napus, which is flanked by donor allele A at position 220 SEQ ID NO: 1 and donor allele A at position 101 SEQ ID NO: 89, and a method for producing Brassica napus meal with low fibre content, comprising identifying a plant sample by this method; method of producing a Brassica napus plant containing low fibre content, comprising recovering or obtaining a nucleic acid sample from each of one or more Brassica napus plants or idioplasms thereof; screening each sample and collecting the first Brassica napus plant or its idioplasm; then crossing the first plant with the second plant to produce descendant plants, where descendant plants contain one or more marker alleles associated with low fibre content, and a method of producing Brassica napus cake with low fibre content, involving obtaining a Brassica napus plant with a low fibre content feature, obtaining seeds from the Brassica napus plant and producing a seed cake.

EFFECT: inventions provide a wider range of methods for reducing fibre content in canola.

15 cl, 1 dwg, 4 tbl, 3 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62/782,699, filed December 20, 2018, the entire contents of which are incorporated herein by reference.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] The present invention relates to detailed mapping of quantitative trait loci (QTL) associated with essential nutritional traits in canola (Brassica napus), including low fiber content. Additional embodiments relate to compositions and methods for identifying a low fiber content trait in a canola plant using molecular markers closely linked to low fiber content. Additional embodiments relate to compositions and methods for introducing a low fiber content trait into a canola plant using these molecular markers.

LEVEL OF TECHNOLOGY

[0003] Canola (Brassica napus L., 2n=4x=38, AACC), an allotetraploid formed from the diploids B. rapa (2n=2x=20, AA) and B. oleracea (2n=2x=18, CC), is one of the most important vegetable oilseed crops in the world, especially in China, Canada, the European Union and Australia. Canola meal, the fraction of the seed that remains after milling and oil extraction, accounts for approximately 55% of the volume of canola seed.

[0004] Canola meal consists of several components including protein, fiber, residual oil, carbohydrates, and antinutritional factors. Although canola meal has a relatively high protein content, its high fiber content reduces its digestibility and value as an animal feed. Compared to soybean meal, canola meal contains high values of dietary fiber and a lower percentage of protein. Because of its high dietary fiber content, canola meal has approximately 20% less metabolizable energy (ME) than soybean meal. As a result, the value of the meal remains low relative to other oilseed meals such as soybean meal, particularly in swine and poultry diets. Rakow (2004a) Canola meal quality improvement through the breeding of yellow-seeded varieties-an historical perspective in AAFC Sustainable Production Systems Bulletin. In addition, the presence of glucosinolates in some types of canola meal also reduces its value due to the detrimental effects of these compounds on the growth and reproduction of livestock.

[0005] Canola varieties differ, in part, in the color of their seed coats. Seed coat colors are generally divided into two main classes: yellow and black (or dark brown). Shades of these colors, such as reddish-brown or yellowish-brown, are also observed. Canola varieties with lighter seed coat colors tend to have thinner skins and therefore less fiber and more oil and protein than varieties with dark 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. Animal Science 62:557–65; Shirzadegan and Röbbelen (1985) Götingen Fette Seifen Anstrichmittel 87:235–7; Simbaya et al. (1995) J. Agr. Food Chem. 43:2062–6; Rakow (2004b) Yellow-seeded Brassica napus canola for the Canadian canola industry in AAFC Sustainable Production Systems Bulletin. One possible explanation for this is that the canola plant can expend more energy on producing proteins and oils if it does not require this energy to produce seed coat fiber components. Yellow-seeded canola lines have also been reported to have lower glucosinolate content than black-seeded canola lines. Rakow et al. (1999b) Proc. 10th Int. Rapeseed Congress, Canberra, Australia, Sep. 26–29, 1999, Poster #9. Thus, yellow-seeded canola varieties have historically been developed as a potential way to increase the nutritional value of canola meal. Bell (1995) Meal and by-product utilization in animal nutrition, in Brassica oilseeds, production and utilization. Eds. Kimber and McGregor, Cab International, Wallingford, Oxon, OX108DE, UK, pp. 301–37; Rakow (2004b), supra; Rakow & Raney (2003).

[0006] Some yellow-seeded forms of Brassica species closely related to B. napus (e.g., B. rapa and B. juncea) have been shown to have lower levels of fiber in their seeds and subsequent meals. Scientists at Agriculture and Agri-Food Canada (AAFC) have developed yellow seed coat (YSC) lines (YN86-37, YN90-1016, YN97-262, and YN01-429) with low skin proportions and thinner seed coats, low fiber, and high oil content compared to black seed coat (BSC) canola (Rakow et al., 2011). In nutritional studies comparing AAFC yellow-seeded canola meal line YN01-429 with B. juncea, B. rapa, and brown-seeded B. napus, the YSC B. napus line was shown to have advantages such as higher protein content, lower fiber content, increased amino acid digestibility and metabolizable energy content, and improved nutrient and energy utilization, based on nutritional status and weight gain in broiler chickens and monogastric species (Hickling, 2009; Slominski et al., 2010).

[0007] Development of a yellow-seeded B. napus germplasm has shown that fiber content can be reduced in B. napus by integrating genes that control seed pigmentation from related Brassica species. However, misconceptions about the inheritance and stability of low-fiber traits and the lack of reliable, high-throughput markers tightly linked to the trait have greatly hampered the breeding of low-fiber traits. Due to allotetraploidy, polymeric gene effects, maternal effects, and environmental influences, the inheritance of the low-fiber trait is complex, and identification of tightly linked markers for the trait is difficult. Current selection of lower fiber canola lines derived from AAFC YSC lines is based primarily on fiber content data obtained through expensive and labor-intensive analytical methods or on seed coat color, due to the close association with low fiber in AAFC YSC lines.

[0008] There is very little information regarding the degree of fiber variability in dark-seeded B. napus germplasm, and there are a few reports of dark-seeded canola lines that have been developed that contain reduced levels of anti-nutritional factors (e.g., fiber and polyphenolic compounds) and increased levels of protein. One such example is open-pollinated varieties (CL044864, CL065620) and hybrids (CL166102H, CL121460H, and CL121466H) of B. napus, which are characterized by favorable seed composition characteristics, including high protein content, low fiber content, reduced polyphenolic content, and increased phosphorus content (U.S. Patent 9,596,871 B2). These desirable nutritional characteristics make this germplasm particularly valuable as sources of canola meal. However, molecular markers that are tightly linked to this essential nutritional trait in dark-seeded canola lines have not been previously described.

BRIEF DESCRIPTION OF THE INVENTION

[0009] Disclosed herein is a method for identifying a quantitative trait locus (QTL) associated with desirable nutritional traits, including low fiber content, in canola. The method comprises obtaining and isolating a nucleic acid sample from a Brassica napus plant or germplasm thereof and screening the sample for a nucleic acid comprising one or more marker alleles associated with low fiber content located within a chromosomal interval on chromosome N13 in Brassica napus. One end of the chromosomal interval N13 is defined by and includes base pair (bp) position 7,301,735 (DBSNP143552; SEQ ID NO:1) and the other end of the N13 interval is defined by and includes bp position 9,417,330 (DBSNP243314; SEQ ID NO:89). For example, a marker allele that is used to screen for a low fiber trait may be one or more marker alleles of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95, or 100. In certain examples, the method includes obtaining a nucleic acid sample from a Brassica napus plant or germplasm and screening the sample against a nucleic acid sample comprising a marker allele associated with low fiber content, SEQ ID NO:90, SEQ ID NO:95, or both SEQ ID NO:90 and SEQ ID NO:95. In another example, the method may include screening a sample for a nucleic acid comprising one or more marker alleles associated with low fiber content located in a smaller chromosomal interval on chromosome N13 in Brassica napus such that one end of the N13 interval is identified by and includes base pair position 8,978,949 (DBSNP02056, SEQ ID NO:61) and the other end of the interval is identified by and includes base pair position 9,375,623 (DBSNP243323, SEQ ID NO:77). Thus, the method may comprise screening a smaller interval for one or more marker alleles of the low fiber trait of SEQ ID NO:61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 100. See Examples 1-3 herein, including Table 3, for additional information regarding the N13 intervals and markers that are used in the disclosed methods.

[0010] Each of the above methods can be used to screen for one or more of the disclosed low fiber marker alleles of chromosome N13 from Brassica napus line CL044864. Each of the above disclosed methods can be used to screen for one or more of the disclosed low fiber marker alleles of chromosome N13 from Brassica napus line CL065620 or derivatives thereof. Screening for the presence of one or more low fiber marker alleles according to the methods disclosed herein can be performed using techniques such as allele-specific polymerase chain reaction (PCR) amplification or nucleic acid sequencing.

[0011] In a specific example, a disclosed method for identifying a plant or germplasm thereof comprises obtaining and isolating a nucleic acid sample from a Brassica napus plant or germplasm and screening the sample for a nucleic acid that comprises one or more low fiber marker alleles of chromosome N13 with a nucleic acid probe comprising SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO 96, or SEQ ID NO:97, or a combination of the foregoing probes. For example, see Example 3 herein.

[0012] The present invention provides a seed of a Brassica napus plant in which the presence of one or more marker alleles associated with low fiber content of chromosome N13, disclosed herein, is identified using the method disclosed herein. The present invention further provides a meal obtained from such seed of a plant in which the presence of one or more marker alleles associated with low fiber content of chromosome N13 is identified.

[0013] Also provided herein is a method of selecting one or more plants or germplasm thereof from a population, wherein the selected plant comprises a quantitative trait locus (QTL) associated with desirable nutritional traits, including low fiber content in canola. The method comprises obtaining and isolating a nucleic acid sample from each plant or germplasm thereof among a plurality of plants in a population of Brassica napus plants, screening each sample for a nucleic acid comprising one or more marker alleles associated with low fiber content in chromosome interval N13 in Brassica napus, which is designated from bp position 7,301,735 (DBSNP143552; SEQ ID NO:1) to bp position 9,417,330 (DBSNP243314; SEQ ID NO:89) and comprising them, wherein the one or more marker alleles are indicative of low fiber content in Brassica napus. The present method includes subsequent selection of one or more plants or germplasm thereof from a population in which the presence of one or more marker alleles associated with low fiber content is identified at the screening stage. For example, one or more marker alleles used to screen for the low fiber trait may be one or more marker alleles of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95 or 100; and each of the selected one or more plants or germplasm thereof contains the above-mentioned one or more marker alleles for which screening was carried out. In certain examples, a method of selecting comprises obtaining a nucleic acid sample from a Brassica napus plant or germplasm and screening the sample against a nucleic acid sample comprising a low fiber marker allele of SEQ ID NO:90, SEQ ID NO:95, or both SEQ ID NO:90 and SEQ ID NO:95; and the one or more plants or germplasm selected comprising one or both of the low fiber marker alleles screened. In one aspect, a disclosed method of selecting may comprise screening each sample against a nucleic acid comprising one or more low fiber marker alleles within the minor chromosomal interval N13 that is designated by bp position 8978949 (DBSNP02056, SEQ ID NO:61) and includes it to the other end that is designated by bp position 8978949 (DBSNP02056, SEQ ID NO:61). 9375623 (DBSNP243323, SEQ ID NO:77) and includes it; and the method includes selecting one or more plants or germplasm thereof characterized by one or more marker alleles associated with low fiber content that have been screened. For example, the selection method may include screening a smaller interval for one or more marker alleles associated with low fiber content under SEQ ID NOs:61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 100; and each of the selected one or more plants or germplasm thereof has one or more marker alleles associated with low fiber content under SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 100, which were screened.

[0014] In each of the above disclosed selection methods, the one or more low fiber marker alleles of chromosome N13 that are screened for and found in each selected plant or germplasm thereof may be one or more marker alleles from Brassica napus line CL044864. Additionally or alternatively, in each of the above disclosed selection methods, the one or more low fiber marker alleles of chromosome N13 that are screened for and found in each selected plant or germplasm thereof may be one or more marker alleles from Brassica napus line CL065620 or a derivative thereof. Moreover, in the disclosed methods for selecting one or more plants or germplasm thereof from a plurality of plants in a population of Brassica napus, the step of screening each sample for a nucleic acid comprising one or more marker alleles associated with low fiber content may be performed using techniques such as allele-specific polymerase chain reaction (PCR) amplification or nucleic acid sequencing.

[0015] In a specific example of a disclosed method for selecting a Brassica napus plant or germplasm, the method comprises obtaining or isolating a nucleic acid sample from a Brassica napus plant or germplasm thereof, screening each sample for a nucleic acid with one or more marker alleles associated with low fiber content, chromosome N13, using a nucleic acid probe comprising SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO 96, or SEQ ID NO:97, or a combination of the foregoing probes.

[0016] The present disclosure also provides seed of a Brassica napus plant selected to have one or more marker alleles associated with low fiber content of chromosome N13 according to any of the plant selection methods described herein. The present disclosure further provides meal from such seed of a plant selected to have one or more marker alleles associated with low fiber content of chromosome N13.

[0017] In another embodiment, disclosed herein is a method of producing a canola plant or germplasm that comprises a quantitative trait locus (QTL) associated with desirable nutritional traits, including low fiber content in canola. The method comprises obtaining and isolating a nucleic acid sample from each of one or more Brassica napus plants or germplasm thereof, screening each sample for a nucleic acid comprising one or more marker alleles associated with low fiber content located within chromosome interval N13 in Brassica napus that is designated from bp position 7,301,735 (DBSNP143552; SEQ ID NO:1) to bp position 9,417,330 (DBSNP243314; SEQ ID NO:89) and comprising them, wherein the one or more marker alleles are indicative of low fiber content in Brassica napus. The method further comprises selecting a first Brassica napus plant in which the presence of one or more marker alleles associated with low fiber content, for which screening was performed, is identified at the screening stage, and then crossing the selected first plant with a second plant to obtain progeny plants, where at least one of the progeny plants contains one or more marker alleles associated with low fiber content, for which screening was performed. For example, the one or more marker alleles used to screen for the low fiber trait may be one or more marker alleles of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95 or 100; so that the selected first plant and at least one progeny plant contain the above-mentioned one or more marker alleles associated with low fiber content, for which screening was carried out. In certain examples of the disclosed method of producing a canola plant, the method comprises screening each sample for a nucleic acid sample comprising a low fiber marker allele of SEQ ID NO:90, SEQ ID NO:95, or both SEQ ID NO:90 and SEQ ID NO:95; such that the selected first plant and at least one progeny plant comprise one or both of the low fiber marker alleles screened for. In one aspect, the disclosed method of producing a canola plant comprises screening each sample for a nucleic acid sample comprising one or more low fiber marker alleles located within and including the minor chromosomal interval N13 that is designated from base pair position 8,978,949 (DBSNP02056, SEQ ID NO:61) to base pair position 9,375,623 (DBSNP243323, SEQ ID NO:77); and the selected first Brassica napus plant and at least one progeny plant comprise one or more desired low fiber marker alleles in the smaller interval of N13. For example, a method for producing a canola plant may comprise screening the smaller interval for one or more low fiber marker alleles of SEQ ID NO:61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 100; such that the selected first Brassica napus plant and at least one progeny plant are characterized by one or more marker alleles associated with low fiber content, for which screening was carried out, under SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 100.

[0018] In each of the above disclosed methods for producing a canola plant, the one or more low fiber marker alleles of chromosome N13 that are screened for and found in the first Brassica napus plant and at least one progeny plant may be one or more marker alleles from Brassica napus line CL044864. Additionally or alternatively, in each of the above disclosed methods for producing a canola plant, the one or more low fiber marker alleles of chromosome N13 that are screened for and found in the first Brassica napus plant and at least one progeny plant may be one or more marker alleles from Brassica napus line CL065620 or derivatives thereof. Moreover, in the disclosed methods for producing a canola plant, the step of screening each sample for a nucleic acid comprising one or more marker alleles associated with low fiber content can be performed using techniques such as allele-specific polymerase chain reaction (PCR) amplification or nucleic acid sequencing.

[0019] In a specific example, a disclosed method for producing canola includes obtaining and isolating a nucleic acid sample from each of one or more Brassica napus plants or germplasm thereof, screening each sample for a nucleic acid comprising one or more low fiber marker alleles of chromosome N13 with a nucleic acid probe comprising SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO 96, or SEQ ID NO:97, or a combination of the foregoing probes.

[0020] The present invention also provides seed from progeny plants of Brassica napus that contain one or more marker alleles associated with low fiber content of chromosome N13 and that are obtained according to any of the methods for producing a canola plant with a trait representing low fiber content described herein. The present invention further provides for the production of meal from such seed of plants with one or more marker alleles associated with low fiber content of chromosome N13.

[0021] Also described are methods for producing a canola plant or germplasm that include a low fiber content trait. Such methods may include introgressing at least one marker allele associated with low fiber content from a first canola plant into a second canola plant, thereby producing a progeny canola plant or germplasm thereof with a marker that represents low fiber content. The introgressed marker is located in chromosome interval N13, which is designated from base pair position 7,301,735 (DBSNP143552; SEQ ID NO:1) to base pair position 9,417,330 (DBSNP243314; SEQ ID NO:89), and is linked to the low fiber content trait in the first canola plant. For example, the introgressed marker may be located within a minor chromosomal interval N13, which is designated from and includes bp position 8978949 (DBSNP02056, SEQ ID NO:61) to and includes bp position 9375623 (DBSNP243323, SEQ ID NO:77), wherein the marker is linked to a low fiber trait in a first canola plant. The introgression process may include any of the above-disclosed methods herein for identifying, selecting, or producing a Brassica napus plant comprising one or more marker alleles associated with low fiber content located within chromosomal interval N13. In certain examples of the disclosed introgression method, a first plant that comprises a low fiber marker within chromosomal interval N13 is crossed with a second plant that does not comprise the low fiber marker to produce an offspring plant that has a lower fiber content relative to the second plant.

[0022] The above and other features will become more apparent from the following detailed description of certain embodiments.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0023] Fig. 1. Genetic map of the 6.2 cM chromosomal interval on N13 where the QTL associated with low fiber content is located. Position 0.0 of the genetic map shown in Fig. 1 corresponds to 32.4 cM in Table 3.

SEQUENCE LIST

[0024] The 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. Only one strand of each nucleic acid sequence is shown, but the complementary strand is intended to be included by any reference to the strand shown. The following is shown in the accompanying sequence listing.

[0025] SEQ ID NOs:1-90, 95 and 100 are marker sequences linked to a QTL associated with low fiber content located in the chromosomal interval at N13.

[0026] SEQ ID NOs:91-94 are primers and probes for the SNP marker n13:58387757 (SEQ ID NO:90) of the TAQMAN™ assay.

[0027] SEQ ID NOs:96-99 are primers and probes for the SNP marker n13_59498877 (SEQ ID NO:95) of the TAQMAN™ assay.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview of some embodiments

[0028] The present invention provides high throughput single nucleotide polymorphism (SNP) markers and high density genetic maps for detailed mapping and validation of a quantitative trait locus (QTL) underlying the low fiber trait derived from black seed canola (BSC) lines. In specific examples, the BSC lines may be CL044864 and CL065620 and derivatives thereof. The SNP markers are closely linked to the low fiber trait and may be used for marker assisted selection (MAS) for the low fiber trait.

[0029] According to the present invention, these SNP markers can be used to introgresse a low fiber content trait, such as from the BSC sources described above, into agronomically desirable canola species and varieties (e.g., to compensate for the lack of low fiber content in cultivated canola). Producing a canola plant with reduced fiber content, compared to traditional varieties, is desirable for a number of reasons. Thus, the methods disclosed herein can be used in high-throughput, low-cost strategies and processes for constructing and executing low fiber content introgression programs in canola.

[0030] In some aspects, the present invention provides compositions and methods for identifying, selecting, and/or obtaining canola plants with a low fiber content trait from the CL044864 and CL065620 BSC lines and derivatives thereof, as well as canola plants or parts thereof, including but not limited to canola seeds and germplasm, that are identified, selected, and/or obtained using the methods of the present invention. The present invention further provides an assay for detecting a low fiber content trait in a canola plant, plant part, and/or canola germplasm.

[0031] The present invention provides a method of using SNP markers and high-density genetic maps based on the low fiber trait of the CL044864 and CL065620 BSC lines using a wide range of phenotypic data from four dihaploid (DH) populations. The present invention is based, at least in part, on the discovery of a major QTL at N13 that explains 65.9% to 71.5% of the variability in the low fiber trait in the two DH populations. This major QTL at N13 was tested and confirmed to be different from the low fiber-associated QTL of the YN01-429 line at N09 YSC (U.S. Patent Application No. 15/731,561) in the two DH populations.

[0032] The present disclosure also provides canola marker loci and chromosomal interval QTLs that exhibit statistically significant cosegregation with low fiber content (which they therefore also predict and indicate). For example, 92 canola marker loci (SEQ ID NOs:1-90, 95, and 100) are disclosed within a 6.2 cM or 2,115,595 bp interval on chromosome N13 on the proprietary B. napus reference genome, DH12075. The interval can be further defined by its location on chromosome N13 from bp position 7,301,735 to bp position 1,000,000. 9417330 on the B. napus reference genome, DH12075, which contains and is flanked by DBSNP143552 (SEQ ID NO:1) and DBSNP243314 (SEQ ID NO:89). In specific examples, markers in this range can be used for marker-assisted selection for the low fiber trait in BSC lines CL044864 and CL065620 and their derivatives, thereby improving the method for breeding low fiber canola lines.

[0033] The present invention also provides methods for identifying a first canola plant or germplasm that exhibits low fiber content. In some examples, at least one allele of one or more marker loci that are linked (e.g., closely linked) to the low fiber content trait from lines CL044864 or CL065620 or derivatives thereof is detected in the first canola plant or germplasm. In some examples, the marker loci may be selected from the loci in Table 3, including SEQ ID NOs: 1-90, 95, and 100.

II. Terms

[0034] Allotetraploid. As used herein, "allotetraploid" generally refers to a hybrid organism with a chromosome complement four times greater than that of a haploid organism.

[0035] Isolated. "Isolated" is a biological component (such as a nucleic acid or protein) that has been substantially separated, prepared separately, or purified from other biological components in a cell of an organism where the component is naturally present (i.e., other chromosomal and extrachromosomal DNA and RNA and proteins) by subjecting the component to chemical and functional changes. For example, and without limitation, a nucleic acid can be isolated from a chromosome by breaking the chemical bonds that join the nucleic acid to the rest of the DNA in the chromosome. Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification techniques. The term also encompasses nucleic acids and proteins produced by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules, proteins, and peptides.

[0036] Nucleic acid molecule. As used herein, the term "nucleic acid molecule" may refer to a polymeric form of nucleotides, which may include both sense and nonsense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of all of the foregoing. A nucleotide may refer to a ribonucleotide, a deoxyribonucleotide, or a modified form of both types of nucleotides. "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 may include one or both of naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.

[0037] Nucleic acid molecules may be modified by chemical or biochemical means, or they may contain non-naturally occurring or derivatized nucleotide bases, as will be readily apparent to those skilled in the art. Such modifications include, for example, labels, methylation, replacement of one or more naturally occurring nucleotides with analogs, internucleotide modifications (e.g., uncharged linkages: e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.; uncharged linkages: e.g., phosphorothioates, phosphorodithioates, etc.; pendant 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 duplex, triplex, hairpin, circular, and locked conformations.

[0038] Mapping population. As used herein, the term "mapping population" may refer to a population of plants (e.g., a population of canola plants) that is used for genetic mapping. Mapping populations are typically derived from controlled crosses of parental genotypes as well as from two inbred lines. Decisions about parent selection, the mating plan for developing the mapping population, and the type of markers used depend on the gene being mapped, the availability of markers, and the molecular map. The parents of plants within the mapping population should have sufficient variability for the trait(s) of interest at both the nucleic acid sequence and phenotype levels. The variability of the parental nucleic acid sequences is used to monitor recombination events in plants of the mapping population.

[0039] The availability of informative polymorphic markers depends on the quantitative significance of the changes in the nucleic acid sequence. Thus, a particular informative marker may not be identified in a particular cross of parental genotypes, although such markers may exist.

[0040] A "genetic map" is a description of the genetic linkage relationships among loci on one or more chromosomes (or linkage groups) within a particular species that can be determined by analyzing a mapping population. In some examples, a genetic map may be depicted in the form of a diagram or table. The term "genetic mapping" can refer to the process of determining linkage relationships of loci using genetic markers, mapping populations that segregate with respect to the markers, and standard genetic principles of recombination frequency. "Location on a genetic map" refers to the location on a genetic map (relative to surrounding genetic markers on the same linkage group or chromosome) where a particular marker from a given species can be found. In contrast, a "physical map of the genome" refers to absolute distances (e.g., base pair measurements or isolated and overlapping close genetic fragments) between markers from a given species. The physical genome map does not necessarily reflect the actual recombination frequency observed when test crossings of species occur between different points on the physical map.

[0041] Crossing. As used herein, the term "crossing" (or "crossed") refers to the fusion of gametes through pollination to produce offspring (e.g., cells, seeds, and plants). The term includes both sexual crossing (i.e., pollination of one plant by another) and self-reproduction (i.e., self-pollination, such as by pollen and ovules from the same plant).

[0042] Backcrossing. Backcrossing techniques can be used to introduce a nucleic acid sequence into plants. Backcrossing techniques have been widely used for decades to introduce new traits into plants. Jensen, N., Ed. Plant Breeding Methodology, John Wiley & Sons, Inc., 1988. In a standard backcrossing protocol, the original variety of interest (the recurrent parent) is crossed with a second variety (the non-recurrent parent) that carries the gene of interest to be passed on. The resulting offspring are then crossed back to the recurrent parent, and the process is repeated until a plant is obtained where virtually all of the desired morphological and physiological characteristics of the recurrent plant are present in the resulting plant, in addition to the transferred gene from the non-recurrent parent.

[0043] Introgression. As used herein, the term "introgression" refers to the transfer of an allele at a genetic locus into a genetic background. In some embodiments, introgression of a specific allelic form at a locus may occur by transferring the allelic form to at least one offspring through a sexual cross between two parents of the same species, wherein at least one of the parents has the specific allelic form in its genome. The offspring containing the specific allelic form may be repeatedly backcrossed to a line with the desired genetic background. The backcross offspring may be selected for the specific allelic form to produce a new variety, wherein the specific allelic form is fixed in the genetic background. In some embodiments, introgression of a specific allelic form may occur by recombination between two donor genomes (e.g., a fused protoplast), wherein at least one donor genome has a specific allelic form in its genome. Introgression may involve the transfer of a specific allelic form, which may be, for example and without limitation, a selected allelic form of a marker allele; a QTL and/or a transgene. In the present invention, introgression may involve the transfer of one or more disclosed marker alleles associated with low fiber content (e.g., those disclosed in Table 3 herein) into an offspring plant.

[0044] Germplasm. As used herein, the term "germplasm" refers to the genetic material of or from an individual plant or group of plants (e.g., a plant line, cultivar, or family) and a clone derived from a plant or group of plants. Germplasm may be part of an organism or cell, or may be separated (e.g., isolated) from an organism or cell. Germplasm generally provides genetic material with a specific molecular pattern that is the basis for the hereditary characteristics of the plant. As used herein, "germplasm" refers to cells of a particular plant; seeds; tissue of a particular plant (e.g., tissue from which new plants can be grown); and non-seed parts of a particular plant (e.g., leaf, stem, pollen, and cells).

[0045] As used herein, the term "gerdioplasm" is synonymous with the term "genetic material" and can be used to refer to seeds (or other plant material) from which a plant can be developed. A "gerdioplasm bank" can refer to an organized collection of different seeds and other genetic material (where each genotype is uniquely defined) from which a known variety can be cultivated and from which a new variety can be developed. In embodiments, the germplasm used in the method or plant described herein is derived from a canola line or variety. In particular examples, the germplasm is seeds of a canola line or variety. In particular examples, the germplasm is a nucleic acid sample from a canola line or variety.

[0046] Gene. As used herein, the term "gene" (or "genetic element") may refer to a hereditary genomic DNA sequence with functional significance. The term "gene" may also be applied to, for example and without limitation, cDNA and/or mRNA encoded by a hereditary genomic DNA sequence.

[0047] Genotype. As used herein, the term "genotype" refers to the genetic makeup of an individual (or group of individuals) at one or more loci. The genotype of an individual or group of individuals is defined and described by the allelic forms at one or more loci inherited by the individuals from their parents. The term genotype may also be applied to the genetic makeup of an individual at one locus, multiple loci, or all loci in its genome. A "haplotype" is the genotype of an individual at multiple genetic loci. In some examples, the genetic loci described by a haplotype may be physically or genetically linked, i.e., the loci may be located on the same chromosomal segment.

[0048] Elite Line. As used herein, the term "elite line" means any line that has been bred and selected for superior agronomic characteristics. An elite plant is any plant from an elite line.

[0049] Quantitative Trait. As used herein, a "quantitative trait" may refer to a trait or phenotype that is expressed to varying degrees, along a generally continuous gradient, and is often linked to two or more genes and is influenced by the environment.

[0050] Quantitative Trait Locus or QTL. As used herein, a "quantitative trait locus" refers to a segment or region of DNA that contains or is linked to a gene or genes corresponding to a quantitative trait.

[0051] As used herein, the term "QTL interval" may refer to a fragment of DNA that is associated with the gene(s) corresponding to the QTL trait. A QTL interval is typically, but not necessarily, larger than the QTL itself. A QTL interval may contain fragments of DNA that are 5' and/or 3' to the QTL.

[0052] A variety of experimental paradigms have been developed for the identification and analysis of QTL. See, for example, Jansen (1996) Trends Plant Sci. 1:89. Most of the literature on QTL mapping in agricultural species is based on the use of biparental crosses. See Lynch and Walsh (1997) Genetics and Analysis of Quantitative Traits, Sinauer Associates, Sunderland. Typically, these paradigms involve crossing one or more parental pairs, which may be, for example, a single pair derived from two inbred lines, or multiple related and unrelated parents of different inbred lines, each exhibiting different characteristics with respect to the phenotypic trait of interest. Typically, this experimental protocol involves generating 100-300 segregated progeny from a single cross of two divergent inbred lines that, for example, have been selected to maximize phenotypic and molecular marker differences between the lines. The parents and segregated progeny are genotyped for marker loci and scored for one or more quantitative traits (e.g., low fiber content). QTLs are then defined as statistically significant associations between genotypic values and phenotypic variation among the segregated generation.

[0053] Those skilled in the art are aware of a variety of statistical methods for determining whether markers are linked to a QTL (or to another marker), including, 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. van Ooijen and J. Jansen (eds.), Biometrics in Plant breeding: applications of molecular markers, pp. 116–24, CPRO-DLO Netherlands; Jansen (1996) Genetics 142:305–11; and Jansen and Stam (1994) Genetics 136:1447–55).

[0054] Illustrative statistical methods include single-point marker analysis; interval mapping (Lander and Botstein (1989) Genetics 121:185); composite interval mapping; penalized regression analysis; complex lineage analysis; MCMC analysis; MQM analysis (Jansen (1994) Genetics 138:871); HAPLO-IM+ analysis, HAPLO-MQM analysis, and HAPLO-MQM+ analysis; Bayesian MCMC; ridge regression; identity by descent analysis; and Haseman-Elston regression, any of which are suitable with respect to particular embodiments of the present invention. Alternative statistical methods applicable to complex breeding populations that can be used to identify and localize QTL in specific examples are described in U.S. Patent 6,399,855 and International PCT Application Publication No. W00149104 A2. All of these approaches are computationally intensive and are typically performed using computer systems including specialized software. Appropriate statistical packages are available from a variety of general and commercial sources and are known to those skilled in the art.

[0055] Marker. Although specific DNA sequences that encode proteins are generally well conserved within a species, other regions of DNA (e.g., noncoding DNA and introns) tend to develop and accumulate polymorphism and thus may differ between individuals of a species. Genomic variation may have any origin, for example, variation may result from insertions, deletions, duplications, repetitive DNA elements, point mutations, recombinations, and the presence and sequence of mobile elements. Such regions may contain useful molecular genetic markers. In general, any differentially inherited polymorphic trait (including nucleic acid polymorphisms) that segregates among offspring is a potential marker.

[0056] As used herein, the term "marker" and "molecular marker" refer to a nucleic acid or its encoded product (e.g., a protein) that is used as a reference point in defining a linked locus. Thus, a marker may refer to a gene or nucleic acid that can identify plants with a particular allele. A marker can be described as a variation at a given genomic locus. A genetic marker can be a short DNA sequence, such as a sequence that surrounds a single base pair change (a single nucleotide polymorphism or "SNP"), or a long one, such as a microsatellite/simple sequence repeat ("SSR"). A "marker allele" or "marker allelic form" refers to the version of a marker that is specific to 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 complementary to the cloned segment of chromosomal DNA. The term also refers to nucleic acid sequences complementary to genomic marker sequences, such as primers and nucleic acid probes.

[0057] A marker may be described, for example, as a specific genetic element at a specific location in a 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 chromosome), where distances between markers on a chromosome are measured by the frequency of recombination between the markers. A genetic marker may be a variety of known polymorphic markers, such as and in particular: simple sequence repeat (SSR) markers; restriction fragment length polymorphism (RFLP) markers; and single nucleotide polymorphism (SNP) markers. As an example, SSR markers may be derived from genomic or expressed nucleic acids (e.g., expressed sequence tags (ESTs)).

[0058] Additional markers include, for example and without limitation, ESTs; amplified fragment length polymorphism (AFLP) markers (Vos et al. (1995) Nucl. Acids Res. 23:4407; Becker et al. (1995) Mol. Gen. Genet. 249:65; Meksem et al. (1995) Mol. Gen. Genet. 249:74); randomly amplified polymorphic DNA, and isozyme markers. Isozyme markers can be used as genetic markers, for example, to track isozyme markers or other types of markers that are linked to a particular first marker. Isozymes are multiple forms of enzymes that differ from each other with respect to amino acid sequence (and thus with respect to their coding nucleic acid sequences). Some isozymes are enzymes that contain slightly different subunits. Other isozymes are either multimeric or monomeric but have been cleaved from the proenzyme at different sites in the proenzyme amino acid sequence. Isozymes can 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 specific examples.

[0059] "Genetic markers" include alleles that are polymorphic in a population, where the alleles can be detected and distinguished by one or more analytical methods (e.g., RFLP analysis, AFLP analysis, isozyme marker analysis, SNP analysis, and SSR analysis). The term "genetic marker" may also refer to a genetic locus ("marker locus") that can be used as a reference point in identifying a genetically linked locus (e.g., QTL). Such markers may also be referred to as "QTL markers."

[0060] Markers corresponding to genetic polymorphisms between members of a population can be detected by methods known in the art. These include, but are not limited to, nucleic acid sequencing, hybridization methods, amplification methods (e.g., sequence-specific PCR amplification methods), restriction fragment length polymorphism (RFLP) detection, isozyme marker detection, allele-specific hybridization (ASH) detection, amplified variable sequence detection of the plant genome, self-sustaining sequence replication detection, simple sequence repeat (SSR) detection, randomly amplified polymorphic DNA (RAPD) detection, single nucleotide polymorphism (SNP) detection, and/or amplified fragment length polymorphism (AFLP) detection. Thus, in certain examples of the present invention, such known methods can be used to detect the SNP alleles described herein. See, for example, Table 3 below.

[0061] "Marker-assisted selection" (MAS) is a method by which phenotypes are selected based on marker genotypes. Marker-assisted selection involves the use of marker genotypes to identify plants for inclusion in and/or removal from a selection or planting program.

[0062] Molecular marker technologies generally improve the efficiency of plant breeding using MAS. A molecular marker allele that indicates linkage disequilibrium with a desired phenotypic trait (e.g., a QTL) provides a useful tool for selecting for the desired trait in a plant population. Key components of implementing a MAS approach are the creation of a dense (information-rich) genetic map of molecular markers in a plant germplasm; the identification of at least one QTL based on statistical associations between the marker and phenotypic variation; the identification of a set of particularly useful marker alleles based on the results of the QTL analysis; and the application and/or extrapolation of this information to the current germplasm breeding population to enable marker-assisted selection decisions.

[0063] Linkage (Dis)Equilibrium. As used herein, the term "linkage disequilibrium" refers to a situation where two markers segregate independently; i.e., the markers are mixed randomly among the offspring. Markers that exhibit linkage equilibrium are said to be unlinked (regardless of whether they lie on the same chromosome). As used herein, the term "linkage disequilibrium" refers to a situation where two markers segregate non-randomly; i.e., the markers have a recombination frequency of less than 50% (and therefore, by definition, are segregated by less than 50 cM on the same linkage group). In some examples, markers that exhibit linkage disequilibrium are said to be linked.

[0064] Linked, tightly linked, and extremely tightly linked. As used herein, linkage between genes or markers may refer to the phenomenon whereby genes or markers on a chromosome exhibit a measurable probability of being passed on to individuals in the next generation together. Thus, the linkage of one marker to another marker or gene can be measured and/or expressed as a recombination frequency. The closer two markers are to each other, the closer the probability becomes to "1". Thus, the term "linked" may refer to one or more genes or markers that are passed on together with a gene with a probability greater than 0.5 (which is expected for an independent set where the markers/genes are located on different chromosomes). When the presence of a gene changes the phenotype of an individual, the markers linked to the gene are said to be linked to the phenotype. Thus, the term "linked" can refer to the relationship between a marker and a gene or between a marker and a phenotype.

[0065] Relative genetic distance (as measured by the crossing over frequency and measured in centimorgans (cM)) 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 exhibit a 1% recombination frequency (i.e., crossing over between the two markers occurs once every 100 cell divisions). In general, the closer one marker is to another marker or gene (whether the distance between them is measured as genetic or physical distance), the more closely they are linked. Since chromosomal distance is approximately proportional to the recombination frequency between traits, there is an approximate physical distance that correlates with the recombination frequency. As used herein, the term "linked" may refer to one or more genes or markers that are separated by a genetic distance of less than approximately 50 cM. Thus, "linked" genes or markers may be separated by less than about 45 cM; less than about 40 cM; less than about 35 cM; less than about 30 cM; less than about 25 cM; less than about 20 cM; less than about 15 cM; less than about 10 cM; and less than about 5 cM.

[0066] As used herein, the term "tightly linked" may refer to one or more genes or markers that are located within 35 cM of one another. Thus, 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 11 cM; less than about 10 cM; less than about 9 cM; less than about 8 cM; less than about 7 cM; less than about 6 cM; less than about 5 cM and even smaller genetic distances.

[0067] As used herein, the term "extremely tightly linked" may refer to one or more genes or markers located within about 5.0 cM of each other. Thus, two "extremely tightly linked" genes or markers may be separated from each other 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; less than about 0.5 cM.

[0068] The closer a particular marker is to a gene that encodes a polypeptide that alters a particular phenotype (whether measured as genetic or physical distance), the more closely linked the particular marker is to the phenotype. Based on the above, it should be understood that markers linked to a particular gene or phenotype include those markers that are closely linked and those markers that are extremely closely linked to the gene or phenotype. In some embodiments, the closer a particular marker is to a gene that alters a low fiber phenotype (whether measured as genetic or physical distance), the more closely linked the particular markers are to the low fiber phenotype. Thus, linked, closely linked, and extremely closely linked genetic markers for the low fiber phenotype in canola may be useful for MAS programs to identify low fiber canola varieties (relative to parent varieties and/or at least one specific conventional variety), to identify individual low fiber canola plants, and to incorporate this trait into other canola varieties (e.g., the "AC" genome, such as B. napus) to reduce fiber content.

[0069] Marker set. As used herein, a "set" of markers or probes refers to a specific collection of markers (or data derived therefrom) that can be used to identify individuals comprising a trait of interest. In some embodiments, a set of markers associated with a low fiber phenotype can be used to identify a low fiber canola plant. Data corresponding to the set of markers (or data derived from the use of such markers) can be stored on an electronic medium. While each marker in a group of markers may have a means with respect to identifying traits, individually selected markers from the group and subgroups, including some, but not all, markers, can also effectively identify individuals with a trait of interest.

[0070] Allele. As used herein, the term "allele" refers to one or more different nucleotide sequences that occur at a particular locus. For example, a first allele may occur on one chromosome, while a second allele may occur on a second homologous chromosome; for example, as well as on different chromosomes of a heterozygous individual, or between different homozygous or heterozygous individuals in a population. In some embodiments, a particular allele at a particular locus may be associated with an agronomically desirable phenotype (e.g., low fiber content). In some embodiments, a particular allele at a locus may allow for the identification of plants without the agronomically desirable phenotype (e.g., high fiber content plants), such that these plants are excluded from a selection or planting program. A marker allele may segregate with a preferred phenotype, thus providing an advantage in identifying plants containing the phenotype. An "allelic form of a chromosomal segment" may refer to a chromosomal segment containing the nucleotide sequence of a marker allele that alters or is associated with a particular phenotype at one or more loci physically located on the chromosomal segment.

[0071] Single Nucleotide Polymorphism. As used herein, the term "single nucleotide polymorphism" (SNP) can refer to DNA sequence variation where a single nucleotide in the genome (or other common sequence) differs between members of a species or between paired chromosomes in an individual. In some examples, markers associated with low fiber content are SNP markers. Modern high-throughput genotyping techniques such as the GoldenGate® and INFINIUM® assays (Illumina, San Diego, CA, USA) can be applied to accurate and rapid genotyping methods using multiplex SNPs from 384-plex to >100,000-plex probes per sample. Other illustrative technologies for studying SNPs include nucleic acid sequencing (e.g., next-generation sequencing or NGS), primer extension, allele-specific PCR (e.g., KASP), H2-dependent PCR (rhPCR), melt-in-mismatch mutation amplification assays (Melt-MAMA), Masscode™ (Qiagen, Germantown, MD, USA), Invader® (Hologic, Madison, WI, USA), sequential signal amplification reaction (SISAR), SnapShot® (Applied Biosystems, Foster City, CA, USA), and Taqman® (Applied Biosystems, Foster City, CA, USA). Although SNP markers are very useful, their discovery requires the availability of high-quality DNA sequence information.

[0072] Plant. As used herein, the term "plant" may refer to a whole plant, a culture of cells or tissues obtained from a plant, and/or any part of any of the foregoing. Thus, the term "plant" includes, for example and in particular, whole plants; plant parts and/or organs (e.g., leaves, stems, and roots); plant tissue; seeds, and a plant cell. A plant cell may be, for example and in particular, a cell in and/or from a plant, a cell isolated from a plant, and a cell obtained by culturing a cell isolated from a plant. Thus, the term "canola plant" may refer to, for example and without limitation, a whole canola plant, multiple canola plants, a canola plant cell(s), a canola plant protoplast, a canola tissue culture (e.g., from which canola can be regenerated), a canola plant callus, parts of a canola plant (e.g., a canola seed, a canola flower, a canola cotyledon, a canola leaf, a canola stem, a canola bud, a canola root, a canola root tip), and canola plant cells, whether whole in a canola plant or in a part of a canola plant.

[0073] Plant line. As used herein, a "line" refers to a group of plants that exhibit low genetic variability (e.g., no genetic variability) between individuals for at least one trait. Inbred lines may be created by multiple generations of selfing 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 refer to a line used for commercial production.

[0074] A "variety" or "cultivar" is a plant line used for commercial production that retains distinct, stable, and consistent characteristics when propagated. In the case of a hybrid variety or cultivar, the parent lines have distinct, stable, and consistent characteristics.

[0075] Commercially suitable. As used herein, the term "commercially suitable" refers to plant lines and hybrids with sufficient plant vigor and fertility such that farmers can produce a crop of the plant line or hybrid using conventional farm equipment. In certain embodiments, commercial plant products with the described components and/or qualities can be extracted from plants or plant materials of the commercially suitable varieties. For example, oil containing desirable fatty components can be extracted from seed of the commercially suitable plant line or hybrid using conventional processing and extraction equipment. In another example, improved canola meal (defined herein) can be produced from ground seed of the commercially suitable plant lines provided by the present invention that contain one or more markers associated with low fiber content disclosed herein. In some embodiments, the commercially suitable plant line is an inbred line or a hybrid line. "Agronomically elite" lines and hybrids typically have desirable agronomic characteristics; for example and without limitation: increased marketable yield, maturity, disease resistance, and lodging resistance of at least one plant.

[0076] Commercial plant product. As used herein, the term "commercial plant product" refers to a raw material obtained from a particular plant or plant part (e.g., a plant containing the germplasm of the present invention and a plant part obtained from a plant containing the germplasm of the present invention). A commercial product may be, for example and in particular: grain; meal; feed; protein; isolated protein; flour; oil; ground or whole grain or seed; any food product that contains any meal, oil; or ground or whole grain; or silage.

[0077] Improved canola meal. As used herein, the term "improved canola meal" means a canola meal obtained from canola seed that is low in fiber and may have increased protein and intrinsic metabolizable energy content and decreased levels of antinutritional factors such as glucosinolates, tannins, phytic acid, sinapine and erucic acid. A meal with some or all of these characteristics may provide an increased input into the diet of animal species, especially monogastric animals. Improved canola meal, which in the present invention may be variously referred to herein as "ECM", "black seed canola ECM", "BSC ECM" or "dark seed canola ECM". The present invention is not limited to black seed canola and dark seed canola ECM.

[0078] Fiber is a component of plant cell walls and includes carbohydrate polymers (e.g., cellulose (linear glucose polymer chains)), hemicellulose (branched chains of heteropolymers, e.g., galactose, xylose, arabinose, rhamnose, with attached phenolic molecules), and pectins (soluble polymers of galacturonic acid, xylose, arabinose, with varying degrees of methylation). Fiber also includes polyphenolic polymers (e.g., lignin-like polymers and condensed tannins).

[0079] Meal quality is measured by the percentages of acid detergent fiber (ADF) and neutral detergent fiber (NDF) in the meal. ADF and NDF levels are critical because they affect animal performance and digestibility. ADF is a measure of the plant components in the least digestible feeds by livestock, including cellulose and lignin. NDF accounts for most of the structural components in plant cells (i.e., lignin, hemicellulose, and cellulose), but not pectin. Lower levels of ADF and NDF also result in a more digestible, high-energy meal.

[0080] In particular embodiments, seed of a canola plant (e.g., a dark-seeded canola plant) that comprises a germplasm described herein may have a reduced level of ADF compared to a reference canola variety. In some embodiments, "high" and "low" component content refers to a comparison between seed obtained from a reference plant comprising a germplasm described herein and seed obtained from standard canola varieties. Thus, a plant that provides seed with a "low" fiber content may provide seed with a lower fiber content than is observed in seed obtained from standard canola varieties.

[0081] Trait or Phenotype. The terms "trait" and "phenotype" are synonymous herein. For purposes of the present invention, traits of particular interest are low fiber content and, in some cases, seed coat color. Some canola varieties exhibit a yellow seed coat color, while other varieties exhibit a dark (e.g., black, dark, and mottled) seed coat color.

[0082] Seed Color. Canola varieties (e.g., inbred canola lines and hybrids) may be characterized by seed color. Canola seed color is typically rated on a scale of 1 to 5 based on seed recovered from healthy plants at or near full seed maturity. "1" indicates a distinct yellow color. "2" indicates generally yellow with brown inclusions. "3" indicates a mixture of brown and yellow. "4" and "5" indicate brown and black, respectively.

III. Mapping and validation of the low fibre trait from CL044864 and CL065620

[0083] Genetic loci corresponding to specific phenotypes, such as low fiber content, can be located in the genome of an organism. By identifying a marker or cluster of markers that co-segregate with the desired trait, a breeder can rapidly select for the desired phenotype by selecting for an appropriate marker (a technique called marker-assisted selection or MAS). Such markers can also be used by breeders to design phenotypes in silico and to implement whole genome selection in practice.

[0084] The present invention provides chromosomal interval and molecular markers associated with low fiber content in canola. The detection of these markers and/or other linked markers can be used to select, identify and/or produce canola plants with low fiber content and/or to remove canola plants from selection programs or from plantings that do not provide for low fiber content.

[0085] The present invention provides a method for identifying and mapping quantitative trait loci (QTL) associated with low fiber content in Brassica napus using single nucleotide polymorphism (SNP) markers. In embodiments, the QTL is identified in BSC lines CL044864 and CL065620. In some embodiments, the markers can be used for marker-assisted selection for a low fiber trait derived from BSC lines CL044864 and CL065620 and derivatives thereof.

[0086] SNP markers and high-density genetic maps were optimized, and fiber content traits were mapped in detail and validated from BSC lines CL044864 and CL065620 with an extensive data set obtained from four dihaploid (DH) populations. These experiments are described in detail in Examples 1-2.

[0087] Table 3 provides the names of 92 markers (SNPs) associated with low fiber content of the present invention, the physical and genetic locations of each marker on canola chromosome N13, and the target alleles associated with low fiber content. The markers of the present invention are described herein relative to the marker loci positions of the DH12075 reference genome in B. napus sequenced at the AAFC through an industry consortium.

[0088] In some examples of the present invention, markers and marker alleles associated with low fiber content, as presented in Table 3, can be located within a chromosomal interval, including, but not limited to (a) a chromosomal interval on chromosome N13 that extends from and includes base pair (bp) position 7,301,735 (DBSNP143552; SEQ ID NO:1) to base pair (bp) position 9,417,330 (DBSNP243314; SEQ ID NO:89); and (b) a chromosomal interval on chromosome N13 that is defined and includes the donor allele for each marker provided in Table 3. In other examples, the marker alleles associated with low fiber content include the markers provided in Table 3 located on a smaller chromosomal interval on chromosome N13 in Brassica napus that is designated from base pair position 8,978,949 (DBSNP02056, SEQ ID NO:61) to base pair position 9,375,623 (DBSNP243323, SEQ ID NO:77). As will be appreciated by one of skill in the art, additional chromosomal intervals can be identified using the SNP markers provided herein in Table 3.

IV. Identification of markers of low fiber content in canola

[0089] Embodiments of the present invention include markers associated with low fiber content, such as in canola derived from BSC lines CL044864 and CL065620 and derivatives thereof. Such markers can be used, for example and without limitation, to identify canola plants or germplasm with an increased likelihood of containing a low fiber phenotype; to select for such canola plants and germplasm (e.g., in a marker-assisted selection program); and to identify and select canola plants and germplasm that do not have a high likelihood of containing a low fiber phenotype. Use of one or more markers described herein can provide advantages in terms of time, cost, and labor required to breed canola for plant breeders compared to currently available compositions and methods in the art. For example, one or more markers described herein may provide improved marker-assisted breeding results for low fiber content in canola compared to currently available markers for this purpose.

[0090] Methods for detecting (identifying) canola plants or germplasm containing specific alleles of low fiber markers are a feature of some embodiments. In some embodiments, any of a variety of marker detection protocols available in the art can be used to detect a marker allele, depending on the type of marker being detected. In examples, suitable methods for detecting markers can include amplification and identification of the resulting amplified marker using, for example and without limitation, PCR, LCR; and transcription-based amplification methods (e.g., SNP detection, SSR detection, RFLP analysis, and many others).

[0091] Typically, a genetic marker relies on one or more properties of nucleic acids to detect it. For example, some techniques for detecting genetic markers utilize hybridization of a nucleic acid probe to a nucleic acid corresponding to the genetic marker (e.g., amplified nucleic acid produced using a canola genomic DNA molecule as a template). Hybridization formats, including, for example and without limitation, liquid phase, solid phase, mixed phase, and in situ hybridization assays, may be suitable for detecting the allele in particular embodiments. Comprehensive instruction on nucleic acid hybridization can be found, for example, in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes Elsevier, NY.

[0092] Markers that correspond to genetic polymorphisms between members of a population can be detected using a variety of 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 methods and sequencing-based methods) can be readily adapted for high-throughput analysis in some examples, for example, using readily available high-throughput methods such as hybridization sequencing.

[0093] Accordingly, the present invention further provides methods for identifying and/or selecting a plant or germplasm having a low fiber content, which comprises: (a) detecting in said plant or germplasm the presence of one or more genetic markers associated with low fiber content in a canola plant, as described herein; and (b) selecting said canola plant or germplasm based on the presence of one or more genetic markers associated with low fiber content in a canola plant.

[0094] Additionally, the methods of the present invention include detecting an amplified DNA fragment associated with the presence of a particular allele of a SNP. In some embodiments, the amplified fragment associated with a particular allele of a SNP has a predicted nucleic acid sequence, and detecting the amplified DNA fragment having the predicted nucleic acid sequence is performed such that the amplified DNA fragment has a nucleic acid sequence that corresponds to (e.g., is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more homologous to) the expected sequence based on the sequence of a marker associated with the SNP in a plant in which the marker was first detected.

[0095] Detection of a particular SNP allele may be accomplished by any of a number of techniques, including, but not limited to, the use of detectable labels. Detectable labels suitable for use include any composition detectable by spectroscopic, radioscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Thus, a particular SNP allele may be detected by, for example, autoradiography, fluorography, or other identical detection techniques, depending on the particular label to be detected. Suitable 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. In some embodiments of the present invention, detection methods include the use of fluorescent dyes.

[0096] Several methods are available for SNP genotyping, including but not limited to hybridization, primer elongation, oligonucleotide ligation, nuclease digestion, minisequencing, and coding spheres. Such methods are reviewed in several publications: Gut, Hum. Mutat. 17:475 (2001); Shi, Clin. Chem. 47:164 (2001); Kwok, Pharmacogenomics 1:95 (2000); 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 employ these and other methods to study SNPs, including Masscode™ (Qiagen, Germantown, MD, USA), Invader® (Hologic, Madison, WI, USA), SnapShot® (Applied Biosystems, Foster City, CA, USA), Taqman® (Applied Biosystems, Foster City, CA, USA), and Infinium Bead Chip™ (Illumina, San Diego, CA, USA). In some embodiments of the present invention, methods for genotyping SNPs include using the Infinium Bead Chip™.

[0097] The SNP markers of the present invention and their corresponding SNP alleles are disclosed in Table 3 and are associated with low fiber content. A single marker or a combination of markers can be used to detect the presence of a plant with low fiber content. For example, a marker can be located within a chromosomal interval that defines a QTL or be present in the plant genome as a haplotype defined herein. A chromosomal interval of the present invention includes a interval on chromosome N13 that is designated from base pair (bp) position 7,301,735 (DBSNP143552; SEQ ID NO:1) to bp position 9,417,330 (DBSNP243314; SEQ ID NO:89) and includes them. A chromosomal interval of the present invention can also be a smaller chromosomal interval on chromosome N13 that is designated from bp position 8978949 (DBSNP02056, SEQ ID NO:61) to position p.o. 9375623 (DBSNP243323, SEQ ID NO:77) and includes them.

[0098] Accordingly, in some aspects of the present invention, there is provided a method of selecting, detecting, and/or identifying a canola plant or germplasm having a low fiber content, the method comprising: detecting in said canola plant or germplasm the presence of a marker (e.g., a marker allele) associated with low fiber content in a canola plant, wherein said marker is located within a chromosomal interval. The chromosomal interval may comprise, consist essentially of, or consist of a chromosomal interval on chromosome N13 that is designated from base position 7,301,735 (DBSNP143552; SEQ ID NO:1) to base position 9,417,330 (DBSNP243314; SEQ ID NO:89) and includes them, thereby identifying and/or selecting the low fiber plants or germplasm. Also, the chromosomal interval may comprise, consist essentially of, or consist of a chromosomal interval on chromosome N13 that is designated from base position 8978949 (DBSNP02056, SEQ ID NO:61) to base position 9375623 (DBSNP243323, SEQ ID NO:77) and includes them. In some examples, each marker described herein may be defined using a donor allele, which may be a donor allele for each marker sequence of SEQ ID NOs:1-89, SEQ ID NO:90, SEQ ID NO:95, and SEQ ID NO:100 described in Table 3.

V. Introgression of markers for low fiber content in canola

[0099] As noted above, identifying canola plants or germplasm that include a marker allele or alleles that are associated with a low fiber phenotype provides a basis for performing canola marker selection. For example, at least one canola plant is selected that includes at least one marker allele that is positively correlated with low fiber content. For comparison, canola plants that include marker alleles that are negatively correlated with low fiber content may be selected.

[0100] Accordingly, the present invention provides methods for selecting canola plants exhibiting low fiber content, comprising detecting in the plant the presence of one or more genetic markers associated with low fiber content as described herein. The present invention provides (a) a method of selecting such a plant, wherein the method comprises obtaining a genomic DNA sample from the canola plant; and (b) detecting the genomic DNA sample of at least one genetic marker associated with low fiber content as described herein. The detecting may comprise detecting one or more SNPs, a combination of SNPs (haplotype), and/or SNPs located within chromosomal intervals that are associated with low fiber content. In some examples of the present invention, the interval is chromosome N13, which is designated from bp position 7,301,735 (DBSNP143552; SEQ ID NO:1) to bp position 9,417,330 (DBSNP243314; SEQ ID NO:89) and includes them, comprising the SNP genetic markers provided as SEQ ID NOs:1-89, SEQ ID NO:90, SEQ ID NO:95, and SEQ ID NO:100, with the donor alleles disclosed in Table 3. In another example of the present invention, the method includes detecting in a genomic DNA sample at least one genetic marker located in the smaller interval N13, which is defined and includes from bp position 8,978,949 (DBSNP02056, SEQ ID NO:61) to bp position 9375623 (DBSNP243323, SEQ ID NO:77), comprising genetic markers (SNPs) provided as SEQ ID NO:61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 100, having the donor alleles disclosed in Table 3.

[0101] The present invention provides a method comprising transferring by introgression a nucleic acid sequence from one donor canola plant with a low fiber content into a recipient canola plant with a high fiber content by crossing the plants. This transfer can be accomplished using conventional crossing techniques. Loci associated with low fiber content are introgressed, in some embodiments, into commercial canola varieties using marker-assisted selection (MAS) or marker-assisted breeding (MAB). MAS and MAB involve the use of one or more molecular markers that have been identified as having a significant probability of co-segregating with a desired trait and are used to identify and select those progeny plants that contain one or more genes encoding the desired trait. As described herein, such identification and selection are based on the selection of one or more SNP alleles located within one of the N13 intervals disclosed herein or one or more markers associated with the SNP alleles. MAB can also be used to develop near isogenic lines (NILs) containing one or more alleles of interest associated with low fiber content, providing a more detailed study of the effect of such allele(s), and is also an effective method for developing populations of backcrossed inbred lines. Canola plants developed according to these embodiments can, in some embodiments, acquire most of their traits from the recipient plant and receive the low fiber trait from the donor plant. MAB/MAS techniques increase the efficiency of backcrossing and introgression genes using marker-assisted selection (MAS) or marker-assisted breeding (MAB).

[0102] Thus, conventional breeding techniques can be used to introgresse a nucleic acid sequence associated with low fiber content into a recipient canola plant with high fiber content. For example, inbred lines of canola plants with low fiber content can be developed using recurrent selection and backcrossing techniques, selfing and/or dihaploids, or any other technique that produces parental lines. In the recurrent selection and backcrossing method, low fiber content can be introgressed into the recipient target plant (the recurrent parent) by crossing the recurrent parent with a first donor plant that is different from the recurrent parent and is referred to herein as the "non-recurrent parent." The recurrent parent is a plant that has a high fiber content and, in some cases, contains 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), etc. In some cases, the non-recurrent parent exhibits low fiber content and contains a nucleic acid sequence associated with low fiber content. The non-recurrent parent may be any plant cultivar or inbred line that is cross-fertile with the recurrent parent.

[0103] In some examples of the disclosed method of introgression, the offspring obtained from a cross between a recurrent and a non-recurrent parent are backcrossed to the recurrent parent. The resulting plant population is then screened for desired characteristics, which can be done in several different ways. For example, the population can be screened using phenotypic pathology selection schemes or quantitative bioassays known in the art. Alternatively, instead of using bioassays, MAB can be performed using one or more of the molecular markers described above herein to identify offspring that contain a nucleic acid sequence associated with low fiber content. MAB can also be used to confirm the result obtained from quantitative bioassays. In some embodiments, the markers defined herein are suitable for selecting suitable offspring plants using genotypic screening.

[0104] Following screening, F1 hybrid plants with a pronounced low fiber phenotype or, in some embodiments, genotype, and thus containing the desired nucleic acid sequence associated with low fiber content, can be selected and backcrossed to the recurrent parent for one or more generations to make the canola plant increasingly inbred. This process can be performed for one, two, three, four, five, six, seven, eight, or more generations.

[0105] Accordingly, the markers of the present invention can be used in MAS methods to identify and/or select and/or produce an offspring with a genetic marker associated with low fiber. Accordingly, the present invention provides a method of selecting a canola plant with low fiber content, the method comprising detecting in a canola germplasm the presence of a marker associated with low fiber content in a canola plant, wherein said marker is located within a chromosomal interval disclosed herein; and selecting a plant from said germplasm, thereby collecting a canola plant with low fiber content. The disclosed chromosomal interval may be interval N13, which is designated from bp position 7301735 (DBSNP143552; SEQ ID NO:1) to bp position 9417330 (DBSNP243314; SEQ ID NO:89) and includes them, including genetic markers (SNPs) provided as SEQ ID NOs:1-89, SEQ ID NO:90, SEQ ID NO:95 and SEQ ID NO:100, having the donor alleles described in Table 3. The interval disclosed by the method can also be the interval N13, which is designated from the position of b.p. 8978949 (DBSNP02056, SEQ ID NO:61) to the position of b.p. 9375623 (DBSNP243323, SEQ ID NO:77) and includes them, including genetic markers (SNPs) provided as SEQ ID NO:61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 100, having the donor alleles described in Table 3.

[0106] The present invention also provides a method for producing a plant and/or germplasm having a low fiber content, 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 a marker associated with low fiber content in a canola plant, wherein said marker is located within a chromosomal interval disclosed herein, harvesting the seed after the crossing, and growing a progeny canola plant from the seed, wherein said progeny canola plant comprises within its genome said marker associated with low fiber content, thereby producing a canola plant having a low canola content. The disclosed chromosomal interval may be interval N13, which is designated from bp position 7301735 (DBSNP143552; SEQ ID NO:1) to bp position 9417330 (DBSNP243314; SEQ ID NO:89) and includes them, including genetic markers (SNPs) provided as SEQ ID NOs:1-89, SEQ ID NO:90, SEQ ID NO:95 and SEQ ID NO:100, having the donor alleles described in Table 3. The interval disclosed by the method can also be the interval N13, which is designated from the position of b.p. 8978949 (DBSNP02056, SEQ ID NO:61) to the position of b.p. 9375623 (DBSNP243323, SEQ ID NO:77) and includes them, including genetic markers (SNPs) provided as SEQ ID NO:61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 100, having the donor alleles described in Table 3.

[0107] In some examples, the second canola plant or germplasm used in the method of the present invention is an elite canola variety. In some examples, by crossing the first and second canola plants, a progeny canola plant or germplasm thereof is obtained that has a low fiber marker that is introgressed into the genome such that it is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% identical to that of the elite canola variety.

[0108] The disclosed method can be used to introgresse a genetic marker associated with low fiber content described herein into a genetic background lacking said marker, wherein the method comprises crossing a donor comprising said marker with a recurrent parent lacking said marker; and backcrossing an offspring comprising said marker with the recurrent parent, wherein said offspring is defined by detecting in its genome the presence of a marker associated with low fiber content in a canola plant. Said marker is located in a chromosomal interval on chromosome N13 that is designated from and includes marker DBSNP143552 (SEQ ID NO:1) at bp 7301735 to marker DBSNP243314 (SEQ ID NO:89) at bp 9417330. In some examples, said marker is located in the N13 interval, which is designated from bp position 8978949 (DBSNP02056, SEQ ID NO:61) to bp position 9375623 (DBSNP243323, SEQ ID NO:77) and includes them. Donor alleles of said markers are defined in Table 3. The method provides a low fiber canola plant or germplasm containing said low fiber associated genetic marker in the genetic background of a recurrent parent, thereby introgressing the low fiber associated genetic marker into a genetic background lacking said marker.

[0109] The present invention provides low fiber canola plants and germplasm. As discussed above, the methods of the present invention can be used to identify, select, and/or obtain a low fiber canola plant or germplasm. In addition to the above methods, the low fiber plant or germplasm may be produced by any method, provided that the marker associated with low fiber content in the canola plant is introduced into the canola plant or germplasm by methods that include, but are not limited to, transformation (including, but not limited to, bacterium-mediated nucleic acid delivery (e.g., via Agrobacterium)), virus-mediated nucleic acid delivery, silicon carbide whisker- or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle bombardment, electroporation, sonication, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical), and/or biological mechanism that results in the introduction of nucleic acid into a plant cell, or any their combination, transformation or fusion of protoplasts, doubled haploid technique, embryo rescue or by any other nucleic acid transfer system.

[0110] "Introducing" in the context of a plant cell, plant, and/or plant part means bringing a nucleic acid into contact with a plant, plant part, and/or plant cell in such a way that the nucleic acid molecule gains access to the interior of the plant cell and/or plant cell and/or plant part. When more than one nucleic acid molecule is introduced, these nucleic acid molecules may be assembled as part of a single polynucleotide construct or nucleic acid construct, or as separate polynucleotide constructs or nucleic acid constructs, and may be located on the same or different nucleic acid constructs. Accordingly, these polynucleotides may be introduced into plant cells by a single transformation, separate transformations, or, for example, as part of a breeding protocol. Thus, the term "transformation" as used herein refers to the introduction of a heterologous nucleic acid into a cell.

[0111] Thus, a canola plant or part thereof with a genetic marker associated with low fiber content that can be obtained using the methods of the subject matter disclosed herein are aspects of the subject matter disclosed herein.

[0112] The canola plant or germplasm may be the progeny of a cross between an elite canola variety and a canola variety comprising an allele associated with low fiber content. In some embodiments, 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 the elite canola variety.

[0113] The canola plant or germplasm may be the offspring of an introgression where the recurrent parent is an elite canola variety and the donor contains a genetic marker associated (e.g., a SNP, a combination of SNPs, SNPs located in a chromosomal interval) with low fiber content in canola, as described herein.

[0114] The canola plant or germplasm may be the progeny of a cross between a first elite canola variety (e.g., a control line) and the progeny of a cross between a second elite canola variety (e.g., a recurrent parent) and a canola variety containing a genetic marker associated with low fiber content in a canola plant as described herein (e.g., a donor).

[0115] Another aspect of the subject matter disclosed herein relates to a method for producing seeds that can be grown into low-fiber canola plants. In some embodiments, the method comprises obtaining a low-fiber canola plant of the present invention, crossing the low-fiber canola plant with another canola plant, and collecting seeds from the cross that, when planted, produce low-fiber canola plants.

[0116] Accordingly, the present invention provides canola plants, seeds, and/or tissue cultures produced by the methods described herein.

[0117] In some embodiments, the subject matter disclosed herein provides methods for analyzing the genomes of canola plants/germplasms to identify those that include desired markers associated with low fiber content. In some embodiments, the methods of analysis include amplifying the sequences of the genomes of canola plants/germplasms and determining nucleotides present at one, more, or all positions of the amplified sequences.

[0118] Accordingly, the present invention provides methods for detecting alleles associated with low fiber content in canola. In some examples, allele recognition is performed in a microtiter plate using Infinium Bead Chip™ technology and a GoldenGate™ PCR-based allele-specific extension assay (Illumina, San Diego, CA, USA), which identifies each SNP with a discrete fluorescent label and a unique targeting address for a specific bead in the array. In further embodiments, reaction products or fluorescent aggregates on the bead are captured and the allele of the SNP associated with low fiber content is determined. In some embodiments, the canola SNP alleles correspond to canola SNP markers comprising the nucleotide sequence of any of SEQ ID NOs: 1-89, SEQ ID NO:90, SEQ ID NO:95, and SEQ ID NO:100.

[0119] All references, including publications, patents, and patent applications, cited herein are hereby incorporated by reference to the extent that they do not conflict with the expressly stated details of the present invention, and are thus incorporated to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in full herein. All references discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the inventors have a right to antedate such invention by virtue of prior invention.

[0120] The following examples are included to demonstrate various embodiments of the present invention and are considered as a detailed catalog of all the various ways in which the present invention can be carried out or all the characteristic parts that can be added to the present invention. Those skilled in the art will appreciate numerous variations, and additions can be made to the various embodiments without departing from the present invention. Accordingly, the following descriptions are intended to illustrate specific embodiments of the present invention, and not to thoroughly specify all permutations, combinations and variations thereof.

EXAMPLES

Example 1. Plant materials

[0121] Two DH populations, PG803 and PG818, were developed from crosses of young canola lines to identify and confirm the low fiber QTL. 363 DH lines of population PG803 were developed from a cross between the black-seeded/high fiber reference line DH12075 and the black-seeded/low fiber NEXERA cultivar CL044864. 367 DH lines of population PG818 were developed from a cross between the black-seeded/low fiber lines NEXERA, CL044864, and CL065620 to confirm that the BSC lines shared the low fiber QTL at N13. Two more populations, PG856 and PG872, were also developed by crossing the low fibre yellow seed YN01-429 from Agriculture and Agri-Food Canada with CL044864 and CL065620, respectively, to validate and confirm that the low fibre QTL found in CL044864 and CL065620 is distinct from the low fibre QTL found in YSC YN01-429. The mapping populations used in this study, their purpose and results are described in Table 1 (BSC=black coated seed; YSC=yellow coated seed; HFC=high fibre; LFC=low fibre; QTL=quantitative trait locus).

Table 1 Pop Female parent Male parent Population size Number of localized markers Target Result PG803 DH12075 (BSC/HFC) CL044864 (BSC/LFC) 363 (DH) 16,216 LFC trait mapping from line BSC CL044864 The LFC QTL from the BSC line CL044864 was identified and mapped in detail to a 6.2 cM region on N13. PG818 CL044864 (BSC/LFC) CL065620 (BSC/LFC) 367 (DH) 1,427 Testing allelism and interaction between two sources of the BSC trait LFC. It was confirmed that CL044864 and CL065620 shared the same LFC QTL on N13. PG856 YN01-429 (YSC/LFC) CL044864 (BSC/LFC) 403 (DH) 3,003 QTL validation. Testing allelism and interaction between BSC and YSC sources of the LFC trait. It was validated and confirmed that the LFC QTLs from the BSC line CL044864 and the YSC line YN01-429 were different. Either the BSC or YSC QTL is sufficient to obtain the LFC trait. PG872 CL065620 (BSC/LFC) YN01-429 (YSC/LFC) 392 (DH) 2,529 QTL validation. Testing allelism and interaction between BSC and YSC sources of the LFC trait. Confirmed QTL LFC from YN01-429 on N09 and QTL LFC from CL065620 on N13. PG2015-1278 DH12075 CL044864 395 (F2) 981 Recombinant selection for detailed mapping of N13. Recombinants selected for further propagation. PG2017-1514 DH12075/
CL044864 DH12075/CL044864 2845 (F2:F3) 48 Detailed mapping of the N13 low fiber locus. The N13 locus was reduced to a region of approximately 400 kb.

[0122] All DH lines from the four populations were grown in the field at Pike Lake, Saskatchewan, Canada during the 2013 and 2014 field seasons. All DH lines were planted in single nursery plots, with each line having a 2-row plot. DH lines were grown and harvested using standard agronomic practices.

Example 2. Phenotypic, genotypic data, construction of a linkage map and localization of QTL.

[0123] Seed harvested from each DH line was cleaned and chemically analyzed for ADF content using the AOAC reference method (AOAC Official Method 973.18). Phenotypic data were collected for two years in 2013 and 2014. Genomic DNA was isolated from the four populations described in Table 1. DH lines derived from all four populations were genotyped using an Illumina Infinium® 60K SNP microarray on a BeadStation 500 G according to the manufacturer's protocol (Illumina, San Diego, CA, USA).

[0124] Individual locations for the four populations DH, PG803, PG818, PG856 and PG872 were constructed using MAPMAKER/EXP 3.0 (Lander et al. 1987; Lincoln et al. 1992) with a LOD score of 10.0 and Haldane cratering function. The consensus map was constructed using Phenomap Enterprise 3.0 (GeneFlow Inc., Centerville, VA, USA). A total of 16,216 SNP markers were localized. Composite interval mapping (CIM) was used to localize QTLs as implemented in QTL Cartographer V2.5 (Wang et al. 2011). A LOD score of 3.0 was used as a threshold to identify genomic regions that significantly affect the acid-detergent fiber content trait.

[0125] A map was constructed for the PG803 population (CL044864) using 363 DH lines and 16,216 SNP markers. One major QTL was identified that explained 71.5% (2013) and 65.9% (2014) of the phenotypic variation in %ADF on chromosome N13. This locus represents the low fiber QTL from the BSC line CL044864.

[0126] A map for the PG818 population (CL044864 x CL065620) was constructed using 367 DH lines and 1427 SNP markers. Table 2 shows exemplary markers obtained by localizing the QTL in PG818: CL044864 x CL065620 to study the allele and interaction of the two QTLs for the LFC trait. One major QTL for low fiber content was identified on chromosome N13 in the same region where it was identified in the PG803 population. Two years of QTL mapping data confirmed that the CL044864 and CL065620 BSC lines harbored the same low fiber content QTL on N13.

[0127] The populations PG856 (YN01-429 x CL044864) and PG872 (CL065620 x YN01-429) were used to study the interaction between the low fiber QTL YSC N09 of YN01-429 and the low fiber QTL BSC N13 of CL044864 and CL065620 and to further validate the BSC N13 QTL. The map for the PG856 population was constructed using 403 DH lines and 3,003 SNP markers. The map for the PG872 population was constructed using 392 DH lines and 2,529 SNP markers. Two QTLs were confirmed at N09 for YSC and N13 for BSC. Ninety-two SNP markers were localized in a 6.2 cM region near the low fiber content QTL on chromosome N13, as shown in Fig. 1 and Table 3.

[0128] This example demonstrates the use of SNP markers and proprietary high-density genetic maps and validation of low ADF content from NEXERA BSC lines CL044864 and CL065620 using two years of phenotypic data from four different DH populations. A major QTL that explains ~70% of the ADF variability was identified on chromosome N13 and validated in two different populations. Markers within this interval near the major ADF content QTL can be used for MAS in canola selection.

[0129] Further detailed mapping narrowed the QTL interval on N13 to a region of approximately 400 kb that was flanked by and included the SNP markers DBSNP02056 (SEQ ID NO:61) and DBSNP243323 (SEQ ID NO:77).

Table 2 Chromium Genetic distance (cM) Number of markers Density Coating N01 4.0 9 0.50 0.02 N02 73.1 96 0.77 0.89 N03 132.5 180 0.74 0.77 N04 50.0 23 2.27 0.97 N05 12.0 17 0.75 0.04 N06 57.9 79 0.74 0.61 N07 85.4 171 0.50 0.99 N08 23.0 24 1.00 0.60 N09 106.4 71 1.52 0.84 N10 54.9 17 3.43 0.43 N11 80.0 102 0.79 0.83 N12 44.3 88 0.51 0.54 N13 146.9 120 1.23 0.98 N14 74.0 31 2.47 0.96 N15 18.3 20 0.96 0.07 N16 75.8 159 0.48 0.74 N17 30.5 8 4.36 0.13 N18 109.9 135 0.82 0.94 N19 121.1 77 1.59 0.99 Total 1300,0 1427 0.92 0.68

Table 3. SEQ ID NO: SNP name Position on the map (cM) Direct SNP Donor allele Physical distance on reference card DH12075 1 DBSNP143552 32.4 [A/G] A N13:7301735..7302454 2 DBSNP251670 32.7 [A/C] A N13:7316677..7316877 3 DBSNP251668 33.2 [T/C] C N13:7321551..7321669 4 BN_N13_7417614 33.8 [G/T] T N13:7417464..7417765 5 BN_N13_7425239 33.8 [A/C] C N13:7425089..7425389 6 BN_N13_7444161 33.8 [G/A] A N13:7444061..7444232 7 BN_N13_7444297 33.8 [A/C] C N13:7444212..7444364 8 DBSNP229192 34.1 [A/C] C N13:7478063..7478364 9 DBSNP00916 34.3 [C/G] G N13:7575499..7576632 10 BN_N13_7600067 34.3 [A/G] G N13:7599917..7600217 11 BN_N13_7612265 34.9 [A/G] G N13:7612115..7612415 12 DBSNP153623 34.9 [T/C] T N13:7623876..7624441 13 BN_N13_7741311 35.2 [T/C] C N13:7741161..7741461 14 BN_N13_7751728 35.2 [T/C] C N13:7751578..7751878 15 BN_N13_7793316 35.2 [A/G] G N13:7793166..7793466 16 BN_N13_7793323 35.2 [A/T] T N13:7793173..7793473 17 DBSNP105065 35.5 [T/C] C N13:7856559..7857277 18 DBSNP106640 36.6 [T/C] T N13:8233323..8234075 19 DBSNP243494 36.8 [A/G] A N13:8256200..8256318 20 BN_N13_8260263 36.8 [A/G] G N13:8260113..8260413 21 DBSNP08830 36.8 [T/G] T N13:8262111..8262320 22 DBSNP243489 36.8 [A/G] G N13:8262292..8262408 23 BN_N13_8262719 36.8 [T/A] A N13:8262569..8262869 24 BN_N13_8262755 36.8 [C/T] T N13:8262605..8362905 25 BN_N13_8262822 36.8 [G/T] T N13:8262672..8262972 26 BN_N13_8264765 36.8 [T/C] C N13:8264615..8264915 27 BN_N13_8271530 36.8 [C/G] G N13:8271380..8271680 28 BN_N13_8271544 36.8 [T/C] C N13:8271394..8271649 29 BN_N13_8271575 36.8 [G/A] A N13:8271425..8271725 95 n13_59498877 36.8 [G/T] T N13:8275781..8276181 30 DBSNP243485 36.8 [A/G] A N13:8300501..8300621 31 DBSNP243479 37.1 [A/G] A N13:8365919..8366215 32 DBSNP03099 37.1 [T/C] C N13:8440074..8440543 33 DBSNP243444 37.1 [T/C] T N13:8654910..8655030 34 DBSNP243439 37.7 [T/C] C N13:8747789..8748089 35 DBSNP25950 38.1 [A/T] A N13:8845683..8845980 36 DBSNP243419 38.1 [T/C] C N13:8865083..8865203 37 DBSNP243415 38.1 [T/C] T N13:8877820..8877939 38 DBSNP243386 38.1 [T/G] T N13:8900069..8900190 39 DBSNP243385 38.1 [A/G] A N13:8900956..8901076 40 DBSNP243383 38.1 [T/C] T N13:8901534..8901657 41 BN_N13_8902116 38.1 [T/C] C N13:8901966..8902266 42 BN_N13_8906796 38.1 [A/G] A N13:8906646..8906946 43 BN_N13_8908503 38.1 [A/T] T N13:8908353..8908653 44 BN_N13_8908557 38.1 [A/T] T N13:8908407..8908707 45 BN_N13_8922223 38.1 [T/C] T N13:8922073..8922373 46 BN_N13_8933300 38.1 [A/G] G N13:8933150..8933450 47 DBSNP243381 38.1 [A/G] G N13:8936136..8936256 48 BN_N13_8956349 38.1 [A/G] A N13:8956199..8956499 49 BN_N13_8956369 38.1 [C/G] G N13;8956219..8956519 50 BN_N13_8956470 38.1 [A/G] G N13:8956320..8956620 51 BN_N13_8956480 38.1 [T/C] T N13:8956330..8956630 52 DBSNP243379 38.1 [A/G] G N13:8956767..8956887 53 DBSNP243378 38.1 [T/C] T N13:8961669..8961790 54 DBSNP324221 38.1 [T/G] T N13:8962358..8963294 55 BN_N13_8965169 38.1 [G/C] C N13:8965019..8965319 56 DBSNP243377 38.1 [A/C] C N13:8965871..8965991 57 DBSNP243376 38.1 [T/G] T N13:8970906..8971026 58 DBSNP243374 38.1 [T/C] T N13:8971421..8971541 59 BN_N13_8976803 38.1 [A/T] A N13:8976653..8976953 60 DBSNP243372 38.1 [T/C] C N13:8977625..8977777 61 DBSNP02056 38.1 [T/C] T N13:8978949..8979328 62 DBSNP90385 38.4 [A/T] T N13:9018526..9018974 63 DBSNP204410 38.4 [T/C] T N13:9028926..9029213 64 DBSNP243362 38.4 [T/G] G N13:9029169..9029469 65 BN_N13_9045055 38.4 [G/A] A N13:9044905..9045205 66 BN_N13_9054936 38.4 [A/G] G N13:9054786..9055086 67 DBSNP243360 38.4 [T/G] G N13:9055229..9055429 68 BN_N13_9055540 38.4 [A/C] C N13:9055390..9055690 69 BN_N13_9057319 38.4 [A/G] A N13:9057169..9057469 70 BN_N13_9057331 38.4 [A/G] G N13:9057181..9057481 100 DBSNP53263 38.5 [A/C] C N13:9086988..9087562 71 BN_N13_9089148 38.5 [T/G] T N13:9088998..9089298 72 BN_N13_9089216 38.5 [C/G] G N13:9089066..9089366 73 BN_N13_9091158 38.5 [A/C] A N13:9091008..9091308 74 BN_N13_9095137 38.5 [A/T] T N13:9094987..9095287 75 BN_N13_9123132 38.5 [A/G] G N13:9122982..9123282 76 BN_N13_9189237 38.5 [G/T] G N13:9189087..9189278 77 DBSNP243323 38.6 [T/C] C N13:9375506..9375623 78 DBSNP243322 38.6 [A/G] G N13:9375578..9375698 79 BN_N13_9376703 38.6 [A/G] A N13:9376553..9376853 80 BN_N13_9378935 38.6 [T/G] G N13:9378785..9379085 81 DBSNP243321 38.6 [A/C] C N13:9379162..9379281 82 DBSNP243319 38.6 [A/C] A N13:9379565..9379685 83 DBSNP243318 38.6 [T/C] T N13:9379638..9379758 90 n13:58387757 38.6 [C/T] T N13:9386901..9387301 84 BN_N13_9388558 38.6 [C/G] G N13:9388408..9388708 85 DBSNP243317 38.6 [A/G] A N13:9389728..9389846 86 DBSNP243316 38.6 [A/G] G N13:9414794..9414914 87 DBSNP38517 38.6 [T/C] T N13:9416209..9417209 88 DBSNP243315 38.6 [T/C] T N13:9416609..9416809 89 DBSNP243314 38.6 [A/T] A N13:9417130..9417330

Example 3: TAQMAN™ Assays

[0130] TAQMAN™ assays were designed for two SNPs that were highly donor specific in the target region: SNP markers n13:58387757 (SEQ ID NO:90) and n13_59498877 (SEQ ID NO:95). The TAQMAN™ primers and probes for both assays are listed in Table 4. The assay mixture used was 1.5 µL of ~6 ng/µL DNA. 18 µM of each probe and 4 µM of each primer were combined to perform each assay. 13.6 µL of the assay was combined with 1000 µL of TOUGHMIX master mix (Quanta Beverly, MA, USA). A MERIDIAN liquid handler (LGC Genomics, Hoddesdon, Hertfordshire, UK) was used to dispense 1.3 μl of the mixture onto a 1536 plate containing ~6 ng of dry DNA. The plate was sealed using a Phusion laser sealer (LGC Genomics, Hoddesdon, Hertfordshire, UK) and thermally cycled using a hydrocycler (LGC Genomics) with the following conditions: 94°C for 15 min, 40 cycles of 94°C for 30 s, 60°C for 1 min. PCR products were measured at 485 (FAM) and 520 (VIC) wavelengths using a Pherastar plate reader (BMG Labtech, Offenburg, Germany). Values were normalized to ROX and plotted and plotted on a scatterplot using KRAKEN software (LGC Genomics, Hoddesdon, Hertfordshire, UK). Genotype was determined by the presence or absence of fluorescent specificity to the SNPs analyzed.

Table 4 SNP name Subsequence Function SEQ ID NO: n13:58387757 CCAAATGAGATTTTC FAM probe 91 CCAAATGAAATTTTC VIC probe 92 TCTAAAGAAACTATGCAATGTTGTAGAGACAAA Direct primer 93 CACAGTTTTTGCTATCTGAGATGTTGT Reverse primer 94 n13_59498877 ATGAGAGCATTCATATTT FAM probe 96 TGAGAGCATGCATATTT VIC probe 97 GCAACATAACTAACAAGTTAAACTCCAATATTCA Direct primer 98 ACGCAACAAAAGCAACGATTAATCA Reverse primer 99

--->

SEQUENCE LIST

<110> Agrigenetics, Inc.

Buyyarapu, Ramesh

Jetty, Raju

Patterson, Thomas G

Preuss, Ryan

Ripley, Van

Rizvi, Masood

Rounsley, Steve

Tahir, Muhammad

<120> SNP MARKERS AND SELECTION FOR LOW FIBER CONTENT

IN REPRESENTATIVES OF THE GENUS BRASSICA

<130> 78760-WO-PCT

<160> 100

<170> PatentIn version 3.5

<210> 1

<211> 520

<212> DNA

<213> Brassica napus

<400> 1

agcttattag cctcaaaaga agttcttcac aagggtctcc ggaagaagat aggaaatgga 60

catacaacga aagtatggga ggagccctgg ctcccaacgc aaccagcgag agcaccactt 120

agcattgaca acgtctgaga cgaagacttc cgcgttcatc atctaattga tgctcagaat 180

cagtcttgga atctagagat actaaacgca gtcatcgccr cggaggatat tcccaggatt 240

acatcactcc gggtgagccg cacaggtcga catgatagtt acttctggga ttttacgaag 300

tccggagtat actcggtgcg atcaggctac aaaagagccc acgagctcca ctctgcggcc 360

aacccgaacg ttgtaacgga acctagtaca acggaactga agaaagcaac atggaagctc 420

aaagccccat gaaaacttaa acactttcta tggcaagtta caacatgata cctagcgacg 480

gcgaagcaac ttaaagtgag acattgtgct aatgaaagta 520

<210> 2

<211> 201

<212> DNA

<213> Brassica napus

<400> 2

gaaggatgat ttattttttg gcgacataag cacactatat aaaggtcaaa acaccattgt 60

tgatctgaaa atcgacagcc actcaagtgt aagtaaatat mtgtagtgat gcatgctttc 120

tgatcttttg caccttaact gtccttaaac catattaccg aaacgtattt atgcaggtgt 180

cgacaaaagt aactgtcaaa a 201

<210> 3

<211> 121

<212> DNA

<213> Brassica napus

<400> 3

atggcattgc atgcgttaca tgaaggtagc gagatgtctg aaaccgtttg caattatcca 60

ytatcgtctt agtgcgagaa tcatgtgtgt tctttagagc tttaggtact atagtaatca 120

g 121

<210> 4

<211> 301

<212> DNA

<213> Brassica napus

<400> 4

ctccgccgtg ggatttgttg aacgatgaga tcatcgatga ggcagcgctt gatcttcgga 60

ccaccacgtg gagctttccg tcgtcaccta tctccgcatc ggtctgatta acaaaacgat 120

gtttttagtc actaggaaat aaaccggaca kaaccaaatt aataaccgaa ccggaagttc 180

aatgccgttt actcgaaatt tctactttgt ccattaatca agctaaatga aagtcaaaat 240

atttctaata agttgatata tatagaaacc caacaacgac atttgattaa ttaagtacta 300

t 301

<210> 5

<211> 301

<212> DNA

<213> Brassica napus

<400> 5

atttattggt tgtgtttggt acttagatta tggagttgta ggggttcacc atgtgggtct 60

gctgtgtgaa aacctagaac ggtcactaga gttttaccag aacattctag gccttgagat 120

caacgaggcg aggccacacg ataagcttcc mtatagagga gcatggttat gggtaggttc 180

agagatgatt catctaatgg agcttccaaa tcctgatcca ttaactggta gacccgagca 240

cggtggtcga gatagacatg cttgtatcgc aatccgagat gtttcagttc tgaaagagat 300

t 301

<210> 6

<211> 172

<212> DNA

<213> Brassica napus

<400> 6

gtgtcttcca cccttctaaa atatctaccg agtcaaaagt aatacgcacg tgatgcatat 60

tttccaccgt attttgactt tgctggtcta aattctaatc rtacgtcgta cgaggagatt 120

taaataattg tacagtggtc ggttggaata ataactggtt tcgaggagca at 172

<210> 7

<211> 192

<212> DNA

<213> Brassica napus

<400> 7

taactggttt cgaggagcaa ttaatcatac attaacatgg agtatgatct aaacattttc 60

tacatccggc attcatactc aaattmtata tttctatgtg aatctaccgt taagaagagt 120

atgtgtgatt ctattataat tgttctaaca aggttttttt tttgaattct ttctaactag 180

gttttataaaac 192

<210> 8

<211> 301

<212> DNA

<213> Brassica napus

<400> 8

attttctttc ttttggcttg cgtaataaca gaaaaaaccc aactttttga tcaataaaaa 60

aaaactgtac ataaagaaat tgcttcaatg ttttatgcaa caacataaac aagtcaagaa 120

gctaaaaaag atcaaagctt tttctttatt magagtttat ttgacgagtt tatccaacaa 180

cataagcaac cggccctaaa cacctcgatg cagcaacaaa aaaaacccac caaaaaaact 240

cagacgatga aaattttttt tgtcagagat tgcatttgag gatttcatca agcaacaaaa 300

a 301

<210> 9

<211> 451

<212> DNA

<213> Brassica napus

<400> 9

tggaagttaa ttttcgcaca aatggaacaa gcgttaaggt taacgttcca gttggtggtg 60

atttgacatt tcacattaac cctgttggcg gactcgtacc acagattgac ggcagattat 120

acgtaaatgg agcccgtgtt tatcctcagg stagcggtgg attcacaata aacgttaacc 180

ctgttgacct gctcgtacct caggctggtg gtggcgtcaa agatgaatat ggatctgaaa 240

accctaaact atctgatcca tctccaggaa tggctaaccc taaggttttc tttgatatga 300

cggtgtgcgg caaaacggtt ggtcggatcg tgatggagct ctttgccgac acgaccccac 360

ggacggcaga gaatttccgc gccctctgta caggcgagaa aggcatgggg aagcttggta 420

agccactcca ttacaaagga tcaatcatcc a 451

<210> 10

<211> 301

<212> DNA

<213> Brassica napus

<400> 10

aacggagagt agtattttat tattgggcct tatgcctcct aaaagtatgt tttactatgg 60

gtctatattt ataacctcac gtacctaaaa gctcttaaaa tgttctttac tataaggccc 120

aagaaatctc actgtcaggt ataaatacat rttatcagct ccaaaaccct aactctgaga 180

ttagtagatt acgcactcct ttaagcttat tgtagccgtg ggaagaaaaa gcagagaagc 240

aaatcagcag ccatgggtaa tcgtcctcat agtttatagt ataatttgcc attagattga 300

a 301

<210> 11

<211> 301

<212> DNA

<213> Brassica napus

<400> 11

ttctacacaa gaaattaaag gaatcaatgg attgaatgct gagcaaaata caggcttttc 60

tttttgatat ctgtccataa ataccggaga taaccgagg aatattttta aaccgtgctt 120

agactaatgg gttgattaag ggcccattta ratcttacta tgcgcgtctt aatttccgta 180

attatatact aattaatttt cccggtctca cgcgttcaaa ataatatccg ttaccacgaa 240

gaatcgtctt cttctctcca cttctcactt tctctttcta aaaaactgct tatttagaga 300

g 301

<210> 12

<211> 450

<212> DNA

<213> Brassica napus

<400> 12

agctttgact cttcacccta tgtgatctcg acacgctgac tcagtcgtta tcgtgcataa 60

gatgtggggg tttcaatgtt taacacttta ctaatattct agtcgcaatc aattcatttt 120

gttggacacg atcgttgcaa ctttttatcy tatttattag tttatttcaa aaatattgtg 180

taactaacac gtatgaaaac ccatgcctat cgttaaatcc tgggcatccc attaattcat 240

tactcctctt tttaacaaac taggtgctga gaccccccgc gcaagcgcag agctggttac 300

tttggatatc ggtggggcgg tataatttac ggaatgttgg tgttgtttaa gatttgtgta 360

aattaaaaaa taaaatttat tggaaataag acaacaaaat tgatcctagc ttttgacgat 420

taactcaagt tagagacatc tcccaaaacg 450

<210> 13

<211> 301

<212> DNA

<213> Brassica napus

<400> 13

cagctctctc aaaggggaac gtggtaataa ttaataaaat ttcttaggtc ttgagacgat 60

aacatattgt tctggtgtca gagagaccgc actttcggat acggtagtgc atggacgtct 120

ctttccacga ttccactcaa aagccaagtt ygttagcttc tcctaggtct acgtagctac 180

atcttatata aaaatgtacc ctaattaata ttttcgttat gtttgtggtc cactaccttc 240

ttcttccttt ttatgattgt tacctctatg gtcttttagg tttaggtcca tagaggaacc 300

t 301

<210> 14

<211> 301

<212> DNA

<213> Brassica napus

<400> 14

tactcttttt accttactat ggttttttcc attgggtttt cctggaaagg tttttaacga 60

ggcaacaaag acgttaagcg agagcggata gtgacaccgg cccccaaggg ggagtgttac 120

gaaagtcaaa gaaaaaaaag cagcagccac ygcaattgtt gaaaatataa aaatgtgggg 180

cccgttgcac tatttgcaca gtaaatttct ttctatatat acgaacgttt tcgttcattt 240

gtaatcgcac atcaaccttc tcctctctct ataataaact cttcctatca gtgttaaaaa 300

t 301

<210> 15

<211> 301

<212> DNA

<213> Brassica napus

<400> 15

tttccatgca taagtcctta tcaggtgttg gagagagttt catgatgaac tctgtgcccc 60

aaggagtgtt ttgatgccca aagaatgtag atgagtcgtt gaaggatcct agcggccagg 120

cgtagcagca aatgctggtc gctacagaag rtatagcaga aaacctgagg cacccaagta 180

ccatagcggg agtgtgtagc gggctagaga gaccgctagt attgaagcgc tttctacagc 240

ggccagccat agcgacttcg cgaggtcgct atgcgttcaa gacttctaaa atctcccaag 300

t 301

<210> 16

<211> 301

<212> DNA

<213> Brassica napus

<400> 16

gcataagtcc ttatcaggtg ttggagagag tttcatgatg aactctgtgc cccaaggagt 60

gttttgatgc ccaaagaatg tagatgagtc gttgaaggat cctagcggcc aggcgtagca 120

gcaaatgctg gtcgctacag aagatatagc wgaaaacctg aggcacccaa gtaccatagc 180

gggagtgtgt agcgggctag agagaccgct agtattgaag cgctttctac agcggccagc 240

catagcgact tcgcgaggtc gctatgcgtt caagacttct aaaatctccc aagtatccta 300

g 301

<210> 17

<211> 519

<212> DNA

<213> Brassica napus

<400> 17

atctgttggg gccttttcta gtgattcaat ttgtttaaat ctctttggtt gcactttgat 60

tatatagttg gaatgaaagc tttgctttat ttggttgttc ttttttagaat tttcagaagt 120

acatagttaa atcctaaagg catgaaaccc tttcggggcag cactctgtct gcatcaacct 180

taaaacacag gacaaccttt aacgacaaca cctttggcyg tcgggcartg cgctagattg 240

caatggtctg aattctcaag cccccaccaa tcaggcaagc ttacgtcatt gttctgaaac 300

acaaagaata tcaacactcc cataatcgcg gtccctgcat caagcgctgc agagaggatg 360

tagttgtgcc tcgcccacca actcttaaac ctcctgaaga tgtagtagtt gaacacaacc 420

ccaacgatgg tccaggacca atagtgcaca gccttggctt gtggcattga gcttactgca 480

gagaagatca atggaatatg gatgtgtttt agccatttc 519

<210> 18

<211> 750

<212> DNA

<213> Brassica napus

<400> 18

atctgttgct gctctttttg tttttttttg gtcaaatttc aggaaatctt agctatgagt 60

atccatatga ttagcaggac tgttcgcaga aattcaatag gagtggcgtt catatgtgca 120

tgtgacaaac gcattggtgc tcaagtcttc cggaacaagc tattcttcat tcttgttagt 180

tcagagatta atcttgtgat gctgttgaat gggttggaga agaaggaatg gaagtgacag 240

tggtttgtcy gcattgccat agcggatgca aatacaaagg caccgctggt tctaggacat 300

ttcacgttat tagctgagtt tgtttttgtg taatatgctt ttattactat aaaaaaagct 360

caatgtttgt ttagtatgct tagaaaacaa tcaaaaactt ttgtgtgttt ggatcttttg 420

tatatttttg tttggataac aatcaaaata tgataatgtc cccaacattt gcacatcaca 480

caaaacaagc tgtcagctaa atcaatgttt acctagtgca tggtagattg atgagcctaa 540

tgcaattgtc accaagggcg tattggttat gctgcatagt gactgagtta agttaagaac 600

caccaaaaac aaatccacaa gagatcaagt taagagacgt tattattgtt ctgttttcat 660

ggtttggaga tgtaattgtt tatcattgac aatggaattt tggaaagaaa aaagcccatt 720

cattatttgg gaattagact tgggacttaa 750

<210> 19

<211> 121

<212> DNA

<213> Brassica napus

<400> 19

ggttttatat aactttartt ttcatgaaat tgtatttcgt tragtttttc gggaaaaatt 60

rtacaaagct attttcagca tcttttatct ttaattaatc gtcgttaagt tmggcatggt 120

g 121

<210> 20

<211> 301

<212> DNA

<213> Brassica napus

<400> 20

atgtggtgta atcttttaca tttcttacct taaaaaggga ttcagtacat ccattaatta 60

catatgccca ttagtctaca aactgaaagt gcaatgaaga agacatatca aataagtaag 120

atgctaagat gcggaggaaa ctgcaacttg rgttgttctt tctttatcct ttgcctccct 180

aagttgcttc cacttctcaa tggaattagt tcccccgttt tgatcaggta tcttaccatc 240

atcaaggacc ttgtgacacc cggcttacca cctcctttta ttttgaatac agcatcacat 300

t 301

<210> 21

<211> 240

<212> DNA

<213> Brassica napus

<400> 21

cgtaggtcgg agtagtatag taggtcctga ttaaatcgcc ttgcgtagaa cggatatgac 60

gaaggtagaa gtagaaaaaa caagtgttag ctcacttggc agcgaaagtt gcagagcgag 120

agagaacaag gttgagaaaa atggcgagca tgtctgcttt ccccgttttc cctctccgct 180

gcttctccgg taactckcat tttcgaatcg cagttctaat ctctcgaaga gtgcctgcag 240

<210> 22

<211> 121

<212> DNA

<213> Brassica napus

<400> 22

agatatcgat ttgttaagga attaaaagtt caatttattc tgrttataac ccmgtaaaga 60

racccattaa acgatgtcgt ttcctattca cgttaaatcg ccttgcgtag aacggrtatg 120

a 121

<210> 23

<211> 301

<212> DNA

<213> Brassica napus

<400> 23

agctcctgtt aaccaaattt catttaacat caacaccagt gtattcctcc taaccaaatc 60

tacaaatcat cctaaaaccc acccaaaaat aaataaaaat tcaataaaag caataataag 120

aaacttaaaa cacacatttt gttttaacaa wagtcttcaa gtgtactgtc catggagtta 180

tctctcctct gattttccga ccgaactgat ctatttccag tctgctcacc accagactcc 240

accgtagggg gaagcggaga aggcgaggca gactttttct gacgttttct ggagtgtctc 300

a 301

<210> 24

<211> 301

<212> DNA

<213> Brassica napus

<400> 24

cagtgtattc ctcctaacca aatctacaaa tcatcctaaa acccacccaa aaataaataa 60

aaattcaata aaagcaataa taagaaactt aaaacacaca ttttgtttta acaatagtct 120

tcaagtgtac tgtccatgga gttatctctc ytctgatttt ccgaccgaac tgatctattt 180

ccagtctgct caccaccaga ctccaccgta gggggaagcg gagaaggcga ggcagacttt 240

ttctgacgtt ttctggagtg tctcacctta atgacacgag tcaaaacctg cacaacgtcc 300

c 301

<210> 25

<211> 301

<212> DNA

<213> Brassica napus

<400> 25

ataaaagcaa taataagaaa cttaaaacac acattttgtt ttaacaatag tcttcaagtg 60

tactgtccat ggagttatct ctcctctgat tttccgaccg aactgatcta tttccagtct 120

gctcaccacc agactccacc gtagggggaa kcggagaagg cgaggcagac tttttctgac 180

gttttctgga gtgtctcacc ttaatgacac gagtcaaaac ctgcacaacg tccctcatgt 240

agggacgttt cctcggtgca cgagagatgc atttataagc aaaagccgca acttcattca 300

c 301

<210> 26

<211> 301

<212> DNA

<213> Brassica napus

<400> 26

aagatataac atcataaatc cgtcaagatt gaatcaacta aacactgtaa aacaagatcc 60

atatatctcc ttagaagaaa agtctaatct ttatcagtca agagacagtt aaaccaagaa 120

tgcaagatgt atgatcatat cagtcaacat ytaacattaa aaacaaacca aacactctaa 180

aaacattata cattccaata atgtataatc ttttatcagc taagagacaa taaacaaaca 240

aaataagaca ttgcagagga aacacagaga caaacctgta agaatattcc aaaatgccag 300

a 301

<210> 27

<211> 301

<212> DNA

<213> Brassica napus

<400> 27

ataatctcac caactcatca tatgaaggta aattggagta atttaattgt gtcaaggatt 60

gatgacagaa aactatatta tccaaatatc cggtttatct tatgaatgca gttgagagtt 120

tggaggttaa taggtgagaa gataaaatgt saaactatat atacttccat atccagttgc 180

aagcagttac aatatgttca cctaaaactc acctaatctc atattcagga ttattagtta 240

tagttcattt tcatccaagg cgacataaat aatgacacca gttcaaaagg acaatatgat 300

g 301

<210> 28

<211> 301

<212> DNA

<213> Brassica napus

<400> 28

tcatcatatg aaggtaaatt ggagtaattt aattgtgtca aggattgatg acagaaaact 60

atattatcca aatatccggt ttatcttatg aatgcagttg agagtttgga ggttaatagg 120

tgagaagata aaatgtcaaa ctatatatac ytccatatcc agttgcaagc agttacaata 180

tgttcaccta aaactcacct aatctcatat tcaggattat tagttatagt tcattttcat 240

ccaaggcgac ataaataatg acaccagttc aaaaggacaa tatgatgttc ttgcttattt 300

c 301

<210> 29

<211> 301

<212> DNA

<213> Brassica napus

<400> 29

attgtgtcaa ggattgatga cagaaaacta tattatccaa atatccggtt tatcttatga 60

atgcagttga gagtttggag gttaataggt gagaagataa aatgtcaaac tatatatact 120

tccatatcca gttgcaagca gttacaatat rttcacctaa aactcaccta atctcatatt 180

caggattatt agttatagtt cattttcatc caaggcgaca taaataatga caccagttca 240

aaaggacaat atgatgttct tgcttatttc tccaccacca ttctaacaat tccttgatac 300

c 301

<210> 30

<211> 121

<212> DNA

<213> Brassica napus

<400> 30

ataatataag tttttaacaa acattagttt gatttcatgt taaactttta ggtttgatat 60

rgttcagctt tcccattaga ctactacggt aaaactaaga atttcttctt ttttytaaag 120

t 121

<210> 31

<211> 301

<212> DNA

<213> Brassica napus

<400> 31

ctaaatttta tatagaatca aatgccatag ggtagtgttt tctgctttgc ttgtaaaatg 60

aaaagtttca gtggaaaatt atttttactt aattatttac aggaaatgat ttggaacgaa 120

taaaatgttt gtattggtcc ctgagaaatt rttttcataa tgctagctac tctagttttg 180

taaagaaaaa tgtacctaat agaccacatg cactatcatt taacattgtc ttcactgtca 240

ctattttcct aataatwaat aacatacaaa gtaaggaaaa aacagatttt tatttattta 300

g 301

<210> 32

<211> 1332

<212> DNA

<213> Brassica napus

<220>

<221> other_feature

<222> (1)..(1)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (2)..(2)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (3)..(3)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (4)..(4)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (5)..(5)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (6)..(6)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (7)..(7)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (8)..(8)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (9)..(9)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (10)..(10)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (11)..(11)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (28)..(28)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (400)..(400)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (821)..(821)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1285)..(1285)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1286)..(1286)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1287)..(1287)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1288)..(1288)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1289)..(1289)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1290)..(1290)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1291)..(1291)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1292)..(1292)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1293)..(1293)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1294)..(1294)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1295)..(1295)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1296)..(1296)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1297)..(1297)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1298)..(1298)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1299)..(1299)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1300)..(1300)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1301)..(1301)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1302)..(1302)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1303)..(1303)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1304)..(1304)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1305)..(1305)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1306)..(1306)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1307)..(1307)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1308)..(1308)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1309)..(1309)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1310)..(1310)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1311)..(1311)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1312)..(1312)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1313)..(1313)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1314)..(1314)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1315)..(1315)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1316)..(1316)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1317)..(1317)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1318)..(1318)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1319)..(1319)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1320)..(1320)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1321)..(1321)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1322)..(1322)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1323)..(1323)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1324)..(1324)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1325)..(1325)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1326)..(1326)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1327)..(1327)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1328)..(1328)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1329)..(1329)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1330)..(1330)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1331)..(1331)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (1332)..(1332)

<223> n is a, c, g, or t

<400> 32

nnnnnnnnnn ncagagaaac aaaagaanaa agaaacattc caagaatatc ttagagattt 60

caagaaaaat gggatcaacg gcggagacac agataactcc ggtacaagtc accgacgacg 120

aagccgctct ctttgccatg cagctagcca gcgcctccgt ccttcccatg gttttaaagt 180

ccgcgctaga ccttgatctt ctcgagatca tggccaagaa ctcttctccg atgtctccct 240

ctgagattgc ttctaaactt cagaccaaaa accccgaagc tccggtcatg ctcgaccgaa 300

tcctccgtct tctcacgtct tactccatcc tcacctgctc caaccgaacc attcccggcg 360

gagacagcgt cgagaggaty tacgggcttg gtccggtttn gcaagtactt gaccaagaac 420

gaagatggtg tctctatagc tgctctttgt cttatgaacc aagacaaggt tctcatggaa 480

agctggtacc atttgaaaga tgcaattctt gatggtggga ttccattcaa caaggcttat 540

ggaatgagcg cttttgagta ccacgggaag gatctaaggt tcaacacggt attcaacaat 600

ggaatgtcta accattcaac cattacaatg aagaagattc tcgagaccta taagggtttt 660

gagggtttga cttctttggt tgacgttggt ggtggcattg gtgctactct caaaatgatt 720

gtctctaagt accctgacct taaaggcatc aactttgatc tcccacatgt catcgaagaa 780

gctacttctc atcccggtat tgatcatgtt ggaggagata ntgtttgtaa gtgtccctaa 840

aggtgatgca attttcatga agtggatatg ccacgactgg agcgatgaac actgcgtgaa 900

attcttgaag aactgctacg aggcgcttcc agaggatgga aaagtgatac tagcagagtg 960

tatacttcca gagacaccag actcaagcct ctcgaccaaa caagtagtcc atgttgattg 1020

cattatgttg gctcacaacc ctggaggcaa agaacggacc gagaaggagt tcgaggcatt 1080

agctaaagga tcaggcttca aaggcatcaa tgttgcctgc aatgcttttg gtgtttacgt 1140

tattgagctg ctcaaaaaga tgtaagacac acacacacac acacaatcca tgtaataatg 1200

atattatatg taaacattgc tttcatgtac gtctacttca ccgtctttgt tttaaaacta 1260

tgatgtgtaa taatggttta ttaannnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1320

nnnnnnnnnn nn 1332

<210> 33

<211> 121

<212> DNA

<213> Brassica napus

<400> 33

tgtagatgtt tataatatta tatgtgaacg ttatatgatc gggtactttt ttctttgaaa 60

yttatatgat cggatactct taacgtaagt acgcatatga ttagcggatc actcacggca 120

g 121

<210> 34

<211> 301

<212> DNA

<213> Brassica napus

<400> 34

atatcgaact ctagaatttt caattcataa aatgaatttt taaatattcg gttatamygt 60

aaattcataa agcataaaat aaatattcat aatatattaa catttttctt acttatacac 120

gatttagttt tggtcattga ctatcaaata yattatgcaa gctattgttt tccatttat 180

tttccattt atgcgagcta ttgttttcca ttttttcatc atctattaat actttttctt 240

tctttaacaa gatttagttt cattccatgt gtcaaacgaa tcatcaagtg cctgytatga 300

a 301

<210> 35

<211> 298

<212> DNA

<213> Brassica napus

<400> 35

aagtcttcca tggtagacct tttccctctg aaactacagt acccacagct cawgcttcca 60

tcatcactgt catcacacat aacccttatt aagtactact aaaaaaaaca aatataaaac 120

attggtccaa agccacattg cctacacaac agtaacgtat atcggaaaat cagtttcttc 180

agtacatgta attaattcaa taaccctaca agctattttc tacaacagag acaaatgaac 240

tcactttttc cagccaccac caatgtaccc gccgtcatca actttctcct tcgggata 298

<210> 36

<211> 121

<212> DNA

<213> Brassica napus

<400> 36

actagccaaa attgattgtt gggcttttgt aagaaacaca ctttattaat tagatataca 60

ygactattaa ctaccatctt tgacctcaaa acctattarc tacaatcttt gacctagaaa 120

c 121

<210> 37

<211> 121

<212> DNA

<213> Brassica napus

<400> 37

gcagtattct tttattgaag agaawattat agttawagtc tcatccatgt caatgtgact 60

yatcaggtat aatatctatg agaaggatga tcaagcatat aacaaaacta gataaccagt 120

m 121

<210> 38

<211> 121

<212> DNA

<213> Brassica napus

<400> 38

ccatatatat kcatgtatgt aagcatttac aacactaact aatgcattgt gtatataaat 60

kggtccctaa attttaacta aaacctaggg agtctaagag catcattatc crgmrtttct 120

t 121

<210> 39

<211> 121

<212> DNA

<213> Brassica napus

<400> 39

gatatgttga gacgccagtc cacatatgca gtaccgaatg taccwagagt tgataaaaaa 60

rggtaatggt tttctgtgcc tcaaagcaac acagtttagc ttagcaatgc aaggctgact 120

a 121

<210> 40

<211> 121

<212> DNA

<213> Brassica napus

<400> 40

tctttgtatg atatgcaacc tcgwtgcaac attttgctcc aaaagctcat gtttatttct 60

ycatttacct gttgtgacct caataaatac taaaagattc gatacaacat gaaaagttar 120

m 121

<210> 41

<211> 301

<212> DNA

<213> Brassica napus

<400> 41

agcattctat tctctaaaag cggttatgtt caatcgtttt aaaacttttt ttttctgaac 60

atgtaataaa taattgaaga tctttatttg tattttatgt ctggtttagg tactaatctt 120

tacgcctata tgcattctac gttctttaat ytaaaagctt attgaatagg ttgtaaaaaa 180

caagaattgt atacgtgact ttgtctatgt aaacttggta ttcaaaactg gttgaccaaa 240

ccgtttgata aatatatgga tatttgctga caagaaaaaa gaccaaggac cataattaac 300

a 301

<210> 42

<211> 301

<212> DNA

<213> Brassica napus

<400> 42

atccaaatgt tacaaccaaa atacagtgaa aaacacaaag catttacata tttaattgaa 60

tttgatcgat atatctcttt caaataaacc acataggtct cactgccaat aacatccgga 120

atcctttaaa caccgcaatt tttctctcta ragtctctcc cctcagcttc cacaaaccct 180

aaatcagccg tcgcatcttt ttcctctggt gaccggtagc tctcttgctc cggtcgccac 240

cccagtccgt catgcgttct agccttcttc cttagcctct ttgtcctacc cttctccttc 300

t 301

<210> 43

<211> 301

<212> DNA

<213> Brassica napus

<400> 43

ttgttgaacc ccaccaatta atatttatca tactcttcgc catttttgat gtttcgggtc 60

atttcacatt tgcttatttc gtatattata aaagaaagca aaactgtaaa caaacttggg 120

aacaaaattc aaactgatat tgttttgttt waaaaaacat ttctattaat ttgcttgatt 180

cttataatag tggtgacatt tagaaccata ataatttgtt cgaacactgt ataactatat 240

atacttacaa aggaccacaa aaatatcttt ctcttcaatt taaaccattc catagtggag 300

a 301

<210> 44

<211> 301

<212> DNA

<213> Brassica napus

<400> 44

cgggtcattt cacatttgct tatttcgtat attataaaag aaagcaaaac tgtaaacaaa 60

cttgggaaca aaattcaaac tgatattgtt ttgtttaaaa aaacatttct attaatttgc 120

ttgattctta taatagtggt gacatttaga wccataataa tttgttcgaa cactgtataa 180

ctatatatac ttacaaagga ccacaaaaat atctttctct tcaatttaaa ccattccata 240

gtggagacaa attcaaaatc aatatatccg taacatcatg cacctaggaa ctgttatccc 300

g 301

<210> 45

<211> 301

<212> DNA

<213> Brassica napus

<400> 45

cactagcagt ttgatattgt cattctggga catccatttt aatcggagca tttacagctg 60

gtacttgtga cttaatcact ttcttatggt cagtaaatga gtctggtaac tcatatgcta 120

atctttgtaa tagaattatc tccatacatt yattttggac ttccatttac gctctttagt 180

ccgaggatca atgataattc attttaactc attcattctc caacgtaact gtttattttc 240

tccctctatt gttggataga ctaactttga gtctttacat aaatcttata aaagcttcta 300

g 301

<210> 46

<211> 301

<212> DNA

<213> Brassica napus

<400> 46

atacccgaaa ccgcacagaa atcactcact cacccatctt cttcatggtg attcttacat 60

agatgagaaa tgattagcac aagacgtttg ttggttgttt aatattcatg cgggatattg 120

tctccttctt cactatttgg taaagctaga rgaaactgtg aaacaaatat actatggtta 180

aattatatat atgatggggc tgattatttg aattggttga gcaggagatt tttttctttg 240

tcaactagca ggggatttac ttatataatt tgtattgtga tgcttgattt acgtgatttc 300

t 301

<210> 47

<211> 121

<212> DNA

<213> Brassica napus

<400> 47

ttagtagcag caagatgcgt ttgtgtatct ttcttgtttc taatgcgact gaattgtatt 60

rtatgctggt aaatggtaac tgtgtgtctg tgttaaagaa atatgcaagg acgctagtct 120

t 121

<210> 48

<211> 301

<212> DNA

<213> Brassica napus

<400> 48

ttatgtcatt gtgaaagtgt agtttatgaa tgtggtaaac atatttacat aaatatctgc 60

ctgcctggtc ttttttgctt gtagttaatg tatgtagcaa acttactctg tcttcttagt 120

ctagattgat attctgaaaa ttaaatttct ractccaaaa tgttaaactg caatttaatt 180

gaagattgca tatatcaaag gtttaaatgg taggatcacc cgcttgcttt cagtgaactg 240

ggttccaagt tatgtattga acaatactag gatcttcacc ccgcgcaagc gcggggatag 300

a 301

<210> 49

<211> 301

<212> DNA

<213> Brassica napus

<400> 49

agtttatgaa tgtggtaaac atatttacat aaatatctgc ctgcctggtc ttttttgctt 60

gtagttaatg tatgtagcaa acttactctg tcttcttagt ctagattgat attctgaaaa 120

ttaaatttct gactccaaaa tgttaaactg saatttaatt gaagattgca tatatcaaag 180

gtttaaatgg taggatcacc cgcttgcttt cagtgaactg ggttccaagt tatgtattga 240

acaatactag gatcttcacc ccgcgcaagc gcggggatag aaggagttcg atttctatgt 300

a 301

<210> 50

<211> 301

<212> DNA

<213> Brassica napus

<400> 50

tagattgata ttctgaaaat taaatttctg actccaaaat gttaaactgc aatttaattg 60

aagattgcat atatcaaagg tttaaatggt aggatcaccc gcttgctttc agtgaactgg 120

gttccaagtt atgtattgaa caatactagg rtcttcaccc cgcgcaagcg cggggataga 180

aggagttcga tttctatgta gtaaacaatt gtgcgggcta tatatatttt gtgtcttatt 240

gttttatggt cccattcat atttatttgc atatttggtt tcataccaca attatctctt 300

g 301

<210> 51

<211> 301

<212> DNA

<213> Brassica napus

<400> 51

ttctgaaaat taaatttctg actccaaaat gttaaactgc aatttaattg aagattgcat 60

atatcaaagg tttaaatggt aggatcaccc gcttgctttc agtgaactgg gttccaagtt 120

atgtattgaa caatactagg atcttcaccc ygcgcaagcg cggggataga aggagttcga 180

tttctatgta gtaaacaatt gtgcgggcta tatatatttt gtgtcttatt gttttatggt 240

cccatttcat atttatttgc atatttggtt tcataccaca attatctctt gaattgtcat 300

t 301

<210> 52

<211> 121

<212> DNA

<213> Brassica napus

<400> 52

aaatcagaac ggaggccgaa gtataagcaa gaaatatgga cgatagtttg aacaaaaaga 60

rcaataaatt atactgaart gttaagtgtt catgacattt gatcatagtt atatgttcat 120

g 121

<210> 53

<211> 121

<212> DNA

<213> Brassica napus

<400> 53

atcactacgg ttcaactttg gcatgctagt gaaccgtagt gatgctagtg atattcccac 60

ygggtcaatt aaccacaaag tgktcatgaa tttctgttct aaggagtttt ggatgcaata 120

c 121

<210> 54

<211> 934

<212> DNA

<213> Brassica napus

<400> 54

aactataaca acgaaaacat aatacatgtt ttggctataa ttgtttgagg gcaccacga 60

actcatatcc taaggtagca aaaagatcgc atcctctagc tcgtggacgg ggtaatcgcg 120

gtggaggtaa agaggtggac gtggataatt tcgtctttga cgttatgaac tctccaagta 180

cttgagtttg agaactctcg gtccttactc tatcccttct tctctattag gaacttgtat 240

tacttctaaa catcagctaa ttagctctcg cttatctcta cttttaaaaa ctgcatgact 300

ttgtcatagc tgtaatggtt acttgcaagt aaatagaaag tttttttttt taaaaaagaa 360

gaagataaac gaagaacatg gttacatgga agttaattaa taagaatgac aaatacatat 420

ttacttagaa aatkaagaag agttcggctc cctgacgttc ttaccttttt gttaaaagat 480

aagttcgtgt tctttccatg acaaaagtat atttggtcca ttacatagtt caattttcct 540

tttgtctcta ctatgaaaga ataagttgtt tattgtgtat gtgtttatac atttagacca 600

aaggttttaa cctcaaaagc tttgacttca aaagctgttt cttttgtgaa tttcaaaaat 660

ttcgaccttc tttcacttta aagtctaaag aacggtgtgc acaaaaaaaa aggggtctaa 720

agaacggttt gctttgtaac tttttttgtt caacacattt tcttttaact ttaatacaaa 780

tgagaatcaa aatattattt ttaataagtt attgattagc ctaacaatgt tttatacttt 840

tcagctttgt ttttaaatgt ttttgggttc aattttaaga tcccattctt gttctagtat 900

tcttgttttc actgcagcaa cgtatgatta tgaa 934

<210> 55

<211> 301

<212> DNA

<213> Brassica napus

<400> 55

tcataaaaac ggcgagcact gcgacgtcac agctcaaacc cgacacgatg ttcttcgatg 60

actctgtttc ctctgtttct tcatcaaaga gattctttga cctcataaag cctctataca 120

acaaaacaac caagaaacag agcgtcaaca stgtatccac atctcctgcg tctttaccgg 180

cgacggcgag ggagaaacag aggaataata aaccgtcagg gattcgaaga cagcttggga 240

agagccggtc ggcgtctgcg actttgtctc cggcgaagag agtcgacgag tctttacagg 300

t 301

<210> 56

<211> 121

<212> DNA

<213> Brassica napus

<400> 56

tctrcaagaa aagccctttc tsaaaaaaga aaaaattcya ccaagaaata tttttrgctc 60

mttactgtga agattaatta tggcatattg tgagatgtgt gacagacgaa gacgacagag 120

t 121

<210> 57

<211> 121

<212> DNA

<213> Brassica napus

<400> 57

aggagyaaaa atatgaatga attaagagct ggtgttttat tcgaaattta atatttaggt 60

kacgaaataa attcgaactt cgactttacy gtacagatta atggattgtg tgaatggtga 120

c 121

<210> 58

<211> 121

<212> DNA

<213> Brassica napus

<400> 58

tacttaaaca ataacttaaa aatatgtagt atatgtcaca tagtcactcg tttaggtcgt 60

ytgttagcgc atgtaatcaa cccaaatata ctattctagt caacaaagga atgaattcca 120

c 121

<210> 59

<211> 301

<212> DNA

<213> Brassica napus

<400> 59

tggacgtctg tggtatcagg gtttgtacga aacttgagag ccaaggaagc agttgctaaa 60

tttcatgaga tgctaggttt gggtttgcaa cccaataact ttacatactt tgcaatcttg 120

atcttgtgtt cctctgtcca gttggtggat wagggcaaga agattcgttc acaggcaata 180

aaggtcgctt ggaggacagc attgatatcg aaaattcact tgtagatatt tagatgaagt 240

gctaggcatc ataaattgtc tgtctcttag acaacgttaa tcttaggtca agcggattac 300

g 301

<210> 60

<211> 201

<212> DNA

<213> Brassica napus

<400> 60

cttatcacat tcttgtgttg tactatgcta ccgagtctgc ccttttcaac ctttttgttc 60

tgagtttgct cattttacaa acactttcat caccgtggag ytgtaatgtc ttttgatttg 120

cgattgcaga atgggaccct tggggtgttc cagatgacta cgagtgtgaa gtaattgaga 180

acgatgcacc cattcccaag c 201

<210> 61

<211> 494

<212> DNA

<213> Brassica napus

<220>

<221> other_feature

<222> (432)..(432)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (468)..(468)

<223> n is a, c, g, or t

<220>

<221> other_feature

<222> (479)..(479)

<223> n is a, c, g, or t

<400> 61

aatgtgtgca aggaagagga agactgggag gcgatcgaga agcgtcttgg ctgcggtcaa 60

gtcgaggagc tcatcgagga ggcgcaagat gagctcacac tcattgcgaa gatgatcgaa 120

tgggaccctt ggggtgttcc agatgactac gagtgtgaag taattgagaa cgatgcaccc 180

attcccaagc acgttcctca gcatcgacct ggtcctcttc ctgaggattt ctacagaacc 240

cttgaaggtc taattacaga gtccaaaaca aaaatcccag ctgccgctac ctccactgat 300

ycgcagttga aggagtgagt aacttccagt tctacattgt ttgtttgtgt tctttgttgc 360

tttgtttggc cactgttcag agacagcgag cctatgaata aactggttaa taatctttga 420

aaactagaac anatatcact cgtttcataa atgtttttat ctttcttnct caaagaacng 480

tatgatata caaa 494

<210> 62

<211> 448

<212> DNA

<213> Brassica napus

<400> 62

aattcgttca aacttttctg gtagtttgga ttgttttaag gtcttacttt tcctatttgg 60

ccgactctgc atgtccagtt tttgtcatta tactatcaag tgaacattac cctggctaag 120

taactaatta acaattctgg tttctgcaac tagagtgttc tttagttttt gcagctttac 180

tacactcttc attttactac tttcttactt atcaaagtat atgataccaa cagcaagtgt 240

gaagaaaaat gatgacactg aaggttgtca gactaagtat aaaccaaaac ccaattctac 300

ttgactagac aaaaatataa tttgcccaaa aacaaactat ttgaacaaga gagataagat 360

ggaagggaaw cattaattta agaacggatg tttgctccaa caacgaaact taactcagat 420

aaaaaaaaca tgaagatagt ccagaatt 448

<210> 63

<211> 301

<212> DNA

<213> Brassica napus

<400> 63

cccgagttga gaactagagt aaggcagtac aaatggtcaa aatcttggcg attcaagttc 60

aaggacaagt caatggaaca cacattatca actggtctct ggtttgtacg gtgataatca 120

gatgcttcta gatgatatag ttsaggaagg ytcagtccta gtggagaagt ttgcctcagt 180

agagatatat aacaaagctc gtttactgct gaagttgatt catatggagt tcattggagg 240

caaagcccgt gacaatgggc tgttagttgc aggtcaaatc aagatttatg tgtgaattct 300

c 301

<210> 64

<211> 301

<212> DNA

<213> Brassica napus

<400> 64

aatcaaatgg tggaagacag tgttgatgat caaaaggtgg ttaaaacaat ggttcaagat 60

attaccgacc aagataaacc tactgaagtt aatgctgtta ctgctagcta ctttgttgac 120

gctgaattgc agttgacaag gaaaagcata kagtagcttc tctaatggtg ttgagatgcc 180

tcaggacttg tttgttatca tgaagctgga ttgtgcagat ccatttcgtt gacactacgt 240

ctccccatga agaattgttg agaactagag taaggcagta caaatggtca aaatcttggc 300

g 301

<210> 65

<211> 301

<212> DNA

<213> Brassica napus

<400> 65

ctctgtagcc gtaaacttcc ctttatctgc cttaagcaac attccgagaa gatcacggtg 60

gtcgtctcca tcgaccaaag atctctttcg ttcgtttatg atcgacaaaa ggagaccatc 120

gatctctttg cctaactctc tagctttgag rgtttgcttg taggccaaaa tgttgctaaa 180

aggtacccct acgtagcgat ttgagttgaa gagagcgaat tgcacggctc ttaggttttt 240

gaggacttga gctccgtttt ctcccttgac tccgaagctt gtcttagcaa tgatctcacc 300

g 301

<210> 66

<211> 301

<212> DNA

<213> Brassica napus

<400> 66

tgtcattata gaatgcaatg atgatggtgt tataatgact taaacaggtt acaatcctcg 60

gaatggctat caagtacggt gggaagtatg tggactcatt tctaaaaggt ataatctctt 120

ccataatttc acacaagagt tgaattacct racgttttgt tggcttattt gagatttgaa 180

acaacaaatg tgcagttttt ttattttcct ggaagcacat tttcaagatc ataatgaatt 240

agtcttccag ctggtaaatt tggtgttctt tgccttttcc tttactgatc tctctgtcca 300

c 301

<210> 67

<211> 201

<212> DNA

<213> Brassica napus

<400> 67

cattcagtaa cgataaagcc attgagattt tgaagagaga ttagtgtgca gtcatgtcgt 60

acccrkccag aagttggagc ckcgtgagtt gtacggagaa kagccttcac gttaaacaga 120

aagcgctctg gagatctctt tgttgctggt attttgctgg tgatggctga ttgwttcmtm 180

ccttaaaaaa aaactcatat a 201

<210> 68

<211> 301

<212> DNA

<213> Brassica napus

<400> 68

tctctcttca aaatctcaat ggctttatcg ttactgaatg ctgcgaacca tttacgtaga 60

ttctcctcta aaattttcac ttccgaaaac ttgaaatcta aatagatccg aaccaaactc 120

gaaaagatat tcaaacgccc acccctgctt mactccaaca acaacaaaac acaccaatgt 180

caaatagcag ataaactagc actgagtgaa tgctcgtata caacctaatg ccgtagaccg 240

tttatataga ttcgagttta cagttataca acaaaatagt atttattttg gttgcaagaa 300

a 301

<210> 69

<211> 301

<212> DNA

<213> Brassica napus

<400> 69

gggacgatct tgaggacgat ttctatgaag agcctaaaag cagcaagaag atgaagaggt 60

ctgatgctac tgcacctaat gatttagatc agaagagcat ccctgaaaag aaacaaggtc 120

caaaggttgt caatttcttt ggatgataca rgaggatcta aaaggtctat caattttgta 180

gggaaatatc agttttttgt tgttctttat ctgcctctag agtttgtgta aggactattt 240

gctatttgga gtttcacaac agtgtcatgt aatggaaccg gtgaaccaca gtcacttctg 300

t 301

<210> 70

<211> 301

<212> DNA

<213> Brassica napus

<400> 70

aggacgattt ctatgaagag cctaaaagca gcaagaagat gaagaggtct gatgctactg 60

cacctaatga tttagatcag aagagcatcc ctgaaaagaa acaaggtcca aaggttgtca 120

atttctttgg atgatacagg aggatctaaa rggtctatca attttgtagg gaaatatcag 180

ttttttgttg ttctttatct gcctctagag tttgtgtaag gactatttgc tatttggagt 240

ttcacaacag tgtcatgtaa tggaaccggt gaaccacagt cacttctgtt tcagataatc 300

t 301

<210> 71

<211> 301

<212> DNA

<213> Brassica napus

<400> 71

cacccaccat ctcaatgtcc attctggttc gaaaaggaaa cagtacatta ctgggaaaga 60

tgagacttat atggaagact cggtccattg tgttccctcg acagagtttg ctggttcgaa 120

gcgcaagcct tcaggggatt tccaacttga kgatccttgg tcatctagag atcatgagat 180

gtttcatttt gaccctgtca ctgagttccc cgatgcacct ctcaaacctt ctgggatcat 240

tcatcctaat gactcttggc catctaaaga tcctgagagg tttgacaaca agtcaggacc 300

t 301

<210> 72

<211> 301

<212> DNA

<213> Brassica napus

<400> 72

atatggaaga ctcggtccat tgtgttccct cgacagagtt tgctggttcg aagcgcaagc 60

cttcagggga tttccaactt gaggatcctt ggtcatctag agatcatgag atgtttcatt 120

ttgaccctgt cactgagttc cccgatgcac stctcaaacc ttctgggatc attcatccta 180

atgactcttg gccatctaaa gatcctgaga ggtttgacaa caagtcagga cctggttctt 240

catcaaagga cacgttctgg gagactgatt ttggagtcga ggataacctt cctggatttg 300

a 301

<210> 73

<211> 301

<212> DNA

<213> Brassica napus

<400> 73

ctctgtttcc gtacctgctg cttttgattc gtctctcatc ttggtgagct tcggaaacct 60

caccttcaag catctgttct ggactttcat cgacaaatct ttgtgcttct cttccacgtt 120

ttttttgtct ctctctctgt tacgtaatct mactctctgc tatctcttgc atttcctcta 180

gatttttgtt ctgcagacga aggttgtttg cttcagccaa tttctttgtt gtcaggttct 240

catgctcgtg atcgatcctc gatctctttc tttaaactcc ttaacctgac tctccagttc 300

a 301

<210> 74

<211> 301

<212> DNA

<213> Brassica napus

<400> 74

tacggtgata atatgttaag tattatttaa ccaagttggt tattgtctgc taatagtttg 60

accattttgg aaccaatcat ttctcagtat gttttcacct aacgttttca ttgaagttta 120

gtaacgtaac tatcacagtt tttgacaaaa wgagactaat gccatcttta agaccttagt 180

taatctgaac tacttaaaac taagcaccta ttgtgaaaag tgaaaacaaa aagagtacga 240

tgatttcact tgtcatggtc tctttataca aaagcaaact caaaaacact atcaatgcca 300

a 301

<210> 75

<211> 301

<212> DNA

<213> Brassica napus

<400> 75

gattacttga cctcttgcct ggaaacatca ggaaacagag ctttgtttgt gtataaatat 60

acatgatcca aagtcctaac tagcccatct ttttaatctt tctggcattt ttcagcttgt 120

tcatgccttt acctgaacat cagttgaact rtcttctctg ttgtatatct gaccaagatt 180

tgatatgttg tttgcaattt gggtagcttc tggagaagga agatcctgca tttcataggt 240

atctttagag ccacttcttg tgatttaata tcaaaagctt cgtgtgagtc actttcatag 300

a 301

<210> 76

<211> 301

<212> DNA

<213> Brassica napus

<400> 76

acaaaataat gatttatatc ggctagtctc aaatcaaata taatttcaga ataaaaaaca 60

aactctactt agttgataac cgaactctaa taccatacat aaatttacat catattttgg 120

aaaaagtaaa agtatatgta taagttttaa kttagctaat tacccttagt tttaagtcaa 180

aactataaaa cagtttttat aaaagctttt ttattttttt gggttttgtt ttatataagg 240

aaaaacaaag aaaaacaaaa caccttcaat cgatattctc tattaaatca aaatttacca 300

c 301

<210> 77

<211> 121

<212> DNA

<213> Brassica napus

<400> 77

ttgggaaata aaaaagatag agtctacaag atgtatgtta tggttgttag ttccgatttg 60

yagagaaagc tcttatgaga aattggagac attctaacaa aagaaccaca aaaatatgca 120

t 121

<210> 78

<211> 121

<212> DNA

<213> Brassica napus

<400> 78

gttatgtata ataataaaac tattctcgtt catagtatca aatttcaaag attatttttg 60

rcttttcatt ttaatttggg aaataaaaaa gatagagtct acaagatgta tgttatggtt 120

g 121

<210> 79

<211> 301

<212> DNA

<213> Brassica napus

<400> 79

acagccacta agctcaagct tcacaagccc cttacaccct tgtgctaata tcgtcaaacc 60

aatatcggaa acagaagaag tatacagtcc ctccacgctt cccaccagtc tcaacactct 120

tagactctcg caagcagcga tgccacgtaa ratattatcg ttacacttgc ggagctcgag 180

ctcttgaaga tcggaacagt gctcggctaa accgagtaaa cctagctcag tagcgttagt 240

caccacgagc ttaagcaaat cgcagcttcc tcttcctaac accataagtc ctctgtcgat 300

t 301

<210> 80

<211> 301

<212> DNA

<213> Brassica napus

<400> 80

ctacaacaca ttccttttgt aagttatatt cgtcgctttc cacatccttt tccggtgact 60

taaactacaa gcctcgacga ggactgaccc ggtggtaaat ggaccttcta tacccacaga 120

ctctggaatt ttcagactcg cgcttgccct kaagttgcta ccgattcttc ttgtgcatcc 180

tccacctgcg tgtgttagac atcctccacc tgatgtctac gatctactat atttaaaata 240

tataagacaa aaaatacagc tcttcaaaat ttacattttg tatcatagac gaaaggcatc 300

c 301

<210> 81

<211> 121

<212> DNA

<213> Brassica napus

<400> 81

wgtacttgga gcttgggtac ggtgaatttt aaacgtttaa gttgtgtaga tgagtgaaac 60

mgaactggtt aagcttagct traaaacttt atatccaatg aatatttcca atattttcaa 120

t 121

<210> 82

<211> 121

<212> DNA

<213> Brassica napus

<400> 82

gataattgtt ggtttgattg tatgatgttt taatacttct ctattcattt atgccaactg 60

mtcatgawtt ttrtaattga aatcaaaagc atamtatacc tktgtttcat acatgactat 120

g 121

<210> 83

<211> 121

<212> DNA

<213> Brassica napus

<400> 83

caaatktrtc aaagtgattg rttttgtatc aaatgtgttg gtataattgt ttggtttgat 60

ytaaattggt ttagataatt gttggtttga ttgtatgatg ttttaatact tctctattca 120

t 121

<210> 84

<211> 301

<212> DNA

<213> Brassica napus

<400> 84

ggttttacgg tgatgtcgtt tcagagagtg aaaagtatcg gtggatgggt cctaaacgct 60

atgattatgt tggaactaaa gtattcttga agcacaggta tttcccttta acatatttac 120

caatttactc ttgccgagat gtgctgaaca stgttgggaa ctatgttaat gcaagatcat 180

atgaggcaga ggtgatgttt gaagaagcag agaacgctaa agcttcaccg ctcacgcgga 240

gcaagacatg gccatttcga agtacaacaa gatcagagaa gatactgtgt cgtgcaaaat 300

g 301

<210> 85

<211> 121

<212> DNA

<213> Brassica napus

<400> 85

aatatccgag tgggacttaa gagataggtt tggrtacaaa accaaatyga atcaaaattc 60

raattaagat ccgaaaattt ccgaaattag cttaatatgt tgatcttttg aagattttaa 120

t 121

<210> 86

<211> 121

<212> DNA

<213> Brassica napus

<400> 86

tatctatctt cagatgcctg ttattattat attacagata atgttatgag taatgaagac 60

rcaatgtgga ctacccctaa aaatggataa actcatatgc ttgtatgtcg ttacaaatgc 120

a 121

<210> 87

<211> 1001

<212> DNA

<213> Brassica napus

<400> 87

aaatcaaaga ctgatgacct ctgccgagga atcttcccgg ccacgacaca ctcttcactc 60

ctgagattag aggagttata cagaggatca gttatctctc tcggttgatg agtctcaaaa 120

gcatccattt tggttatgat gcaaaggaga agagagagag agatgatatt ggtttgaata 180

ttactatgat ggtcatcacg acaacacttg acatttatta tattacagaa aatccctttg 240

acatttgact tcgtagctaa ttaaaagttc cttaaatgaa agaaatggtt acaagtgtca 300

tttgactttt gtagcatttg acttctagtt atctaagtcc agactttctg cctgagatca 360

tgaacgttca attaagaaga gagtttctgt tattatggga aggaagagaa acaaagacac 420

ttaaaacaag actcctaaac ctataattca atctctgtgg aatgaaccct ttcttctagg 480

ggaatgtaac attaaccttt yatcaaaatc atgatgctgt tggaactatc aggcatcttt 540

aaagaccagg aatagcctcc aggttatact tttcttcagc tttttgagct tcgccttccc 600

cactctctgg aggcggtttc ggcttctcca acgcatctag actctggcga tacacatagg 660

aagagctttg ccggttttta ctgctagaat tgcttatttg actagcaaga aaccttcgga 720

tgagccccct cagaaacgga gccaacgtat ctggttcatg ttctaggtaa tacattattc 780

ctcccataca tgcacagaac atagccacct gttttcattg attgtataaa taaaaagatc 840

agttctattg ttaaagagga agaacggttg gtcccttgaa ttgtagtacc tcggcgtttt 900

taatatcagg aagaacgtgg cggttgacaa gtatctccca cagtgaatct cctgctcggg 960

gaagaacgta tagagcaagc tcagagcgtc tcggtttttt c 1001

<210> 88

<211> 201

<212> DNA

<213> Brassica napus

<400> 88

aggaagagaa acaaagacac ttaaaacaag actcctaaac ctataattca atctctgtgg 60

aatgaaccct ttcttctagg ggaatgtaac attaaccttt yatcwaaatc atgatgctgt 120

tggamctatc aggcatcttt aaagaccagg aatagcctcc aggttatact tttcttcagc 180

tttttgagct tcgccttccc c 201

<210> 89

<211> 201

<212> DNA

<213> Brassica napus

<400> 89

ggttgacaag tatctcccac agtgaatctc ctgctcgggg aagaacgtat agagcaagct 60

cagagcgtct cggttttttc tccagcataa ccgagagagc wgctacacca cccgcgaacc 120

agtaaacgat cttgtggtcc ttggwtgcaa cttttctatg kgcacatatg aaagcctatt 180

tgcaaatggg acataaagta t 201

<210> 90

<211> 401

<212> DNA

<213> Brassica napus

<220>

<221> other_feature

<222> (86)..(86)

<223> n is A, T, C or G

<220>

<221> other_feature

<222> (316)..(316)

<223> n is A, T, C or G

<400> 90

gttctttgtt ttggatggat gccattacat caaatgcatg acctgctcgt tctgttacaa 60

tcacctgatt tcaatggtaa atcaanaata tcaaactaag gacagttata gtatgcacag 120

cgagaaaaac acgtatgtct ttctgtatga gagttatatt atctaaagaa actatgcaat 180

gttgtagaga caaagaaaat ytcatttggc caacacaaca tctcagatag caaaaactgt 240

ggatatctac taaaacgcat aactactcac tttgcttcaa atgactacag aataaacaac 300

aaataactat tgatcnggac aaaccccaaa gaagcaaata caataaatgg taataacatt 360

ttcccctaat gctctaccat agcacacaat aataaatact c 401

<210> 91

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide probe

<400> 91

ccaaatgaga ttttc 15

<210> 92

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide probe

<400> 92

ccaaatgaaa ttttc 15

<210> 93

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide primer

<400> 93

tctaaagaaa ctatgcaatg ttgtagagac aaa 33

<210> 94

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide primer

<400> 94

cacagttttt gctatctgag atgttgt 27

<210> 95

<211> 401

<212> DNA

<213> Brassica napus

<220>

<221> other_feature

<222> (108)..(109)

<223> n is A, T, C or G

<220>

<221> other_feature

<222> (111)..(112)

<223> n is A, T, C or G

<220>

<221> other_feature

<222> (169)..(169)

<223> n is A, T, C or G

<220>

<221> other_feature

<222> (239)..(239)

<223> n is A, T, C or G

<400> 95

ggcgccagcc tttgcctgta ccggttaggg tcctgggcac gtctccccgc cctgcgaatc 60

gaacagctga cctccccaag gcgcagatac cactggacta tcgagtcnng nnagtagcaa 120

cataactaac aagttaaact ccaatattca tgataatatg ttactatana gtcgtcgtat 180

gaataagaaa atgagagcat kcatatttgt attattttaa ttagtacaag caaattaana 240

ggagatgatt aatcgttgct tttgttgcgt cgcatgtgtg attacttcaa acgtggcagc 300

attttggata tgtcgtcctc tgtcgttaaa ttagggttta aatggagttg ttttcgtttc 360

atgaatttat tacacatatt acgttggacc ttcatgttct a 401

<210> 96

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide probe

<400> 96

atgagagcat tcatattt 18

<210> 97

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide probe

<400> 97

tgagagcatg catattt 17

<210> 98

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide primer

<400> 98

gcaacataac taacaagtta aactccaata ttca 34

<210> 99

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide primer

<400> 99

acgcaacaaa agcaacgatt aatca 25

<210> 100

<211> 590

<212> DNA

<213> Brassica napus

<400> 100

gaggctattg gtgtccatgg ggttatctta caatgagctc aacagtccgt ccccaagtgg 60

aaatcaactg aatcttaatg aagcaacagm aycttgttcc ctgccaacca cagactgtct 120

gcttgtcgct tacgagtgag tatcatttat tgctataata ttattctctc aatgatcata 180

gcctttgaaa aatgactttt atgggaagtg gacagggaaa atatttgttg atgaataacg 240

gtaggggtct tttattatga tgattgcaag agctagatag tgctaaatac tgttgcttcc 300

caacctctga catattataa tgacatttgc ttgtgtgaat tgcttttttt tatgctcttt 360

ggatcttttc atgctaagaa acataccttt atctattctt gttcaacatt ttcggctaaa 420

ctgttctttt ggagagtagg caagctagct aactagttct tgctatttta ttctaggcgc 480

atttctccat cttttcctgt ggagaatttc agcgtcattg tagaatatgg aggtccaaat 540

gcatcgccca gagtctcatc tcctttaaag ttggactcct ttccttgctt 590

<---

Claims (30)

1. A method for identifying a sample of Brassica napus plant that includes a trait representing low fiber content, the method comprising: a. obtaining a nucleic acid sample from the Brassica napus plant; b. screening the sample for a nucleic acid comprising one or more marker alleles associated with low fibre content located within chromosome interval N13 in Brassica napus that is flanked by and includes the donor allele A at position 220 of SEQ ID NO:1 and the donor allele A at position 101 of SEQ ID NO:89, wherein the marker allele is indicative of low fibre content in Brassica napus. 2. The method of claim 1, wherein the method comprises screening for one or more marker alleles associated with low fiber content under SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95 or 100. 3. The method according to claim 1 or 2, wherein the method comprises screening for one or more marker alleles associated with low fiber content located in chromosome interval N13 in Brassica napus, which is flanked by and includes a T donor allele at position 301 of SEQ ID NO:61 and a C donor allele at position 61 of SEQ ID NO:77. 4. The method of claim 3, wherein the method comprises screening for one or more marker alleles associated with low fiber content under SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 100. 5. The method according to any one of claims 1-4, wherein the Brassica napus plant sample comprises one or more marker alleles associated with low fiber content in SEQ ID NO:90 or SEQ ID NO:95. 6. The method according to any one of claims 1-5, wherein screening for one or more marker alleles associated with low fiber content comprises amplification by allele-specific polymerase chain reaction (PCR) or nucleic acid sequencing. 7. The method according to any one of claims 1-6, wherein screening for one or more marker alleles associated with low fiber content comprises using a nucleic acid probe comprising SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO 96 or SEQ ID NO:97, or a combination of said probes. 8. The method according to any one of paragraphs 1-7, wherein the method includes: a. obtaining nucleic acid samples from each plant among a plurality of plants in a plant population; b. screening each sample for a nucleic acid comprising one or more marker alleles associated with low fibre content according to the method of any one of claims 1 to 7, and c. selecting (i) one or more plants from a population in which the presence of a marker allele associated with low fiber content is identified by screening in step b, or (ii) germplasm from one or more plants from a population in which the presence of a marker allele associated with low fiber content is identified by screening in step b. 9. A method for producing low-fiber Brassica napus meal comprising a. identifying a sample of a Brassica napus plant having the low fibre characteristic, according to any one of paragraphs 1 to 8, wherein the identified sample is obtained from a Brassica napus plant having the low fibre characteristic; b. obtaining seeds from the Brassica napus plant; and c. production of meal from seeds. 10. A method for producing a Brassica napus plant containing a trait that is low in fiber content, the method comprising a. isolating or obtaining a nucleic acid sample from each of one or more Brassica napus plants or their germplasm; b. screening each sample for nucleic acid containing one or more marker alleles associated with low fiber content located within chromosome interval N13 in Brassica napus that is flanked by and includes the donor allele A at position 220 of SEQ ID NO:1 and the donor allele A at position 101 of SEQ ID NO:89; c. selecting the first Brassica napus plant or germplasm thereof in which the presence of one or more marker alleles associated with low fibre content is identified by screening in step b; and d. crossing the first plant selected in step c with a second plant to produce progeny plants, wherein the progeny plants contain one or more marker alleles associated with low fiber content. 11. The method of claim 10, wherein the marker alleles are one or more of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95 or 100. 12. The method of claim 10 or 11, wherein the first Brassica napus plant and the progeny plants comprise a marker associated with low fiber content (i) located in chromosome interval N13 in Brassica napus that is flanked by and includes base pair position 8978949 (DBSNP02056, SEQ ID NO:92) and base pair position 9375623 (SEQ ID NO:77), (ii) from the CL044864 line or derivatives thereof, (iii) from the CL065620 line or derivatives thereof, (iv) SEQ ID NO:61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 100, (v) SEQ ID NO:90 or (vi) SEQ ID NO:95. 13. The method according to any one of claims 10-12, wherein at least one marker allele is detected using sequence-specific amplification based on polymerase chain reaction (PCR). 14. The method according to any one of claims 10-13, wherein at least one marker allele is detected using a nucleic acid probe comprising SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:96, SEQ ID NO:97 or a combination of said probes. 15. A method for producing low-fiber Brassica napus meal comprising a. obtaining a Brassica napus plant having the low fibre content trait according to any of paragraphs 10-14; b. obtaining seeds from the Brassica napus plant; and c. production of meal from seeds.

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