Creation method and application of clubroot composite resistance germplasm

Abstract

According to the method, phenotypic character materials controlled by a single gene and resistance materials identified through indoor inoculation of different clubroot races serve as parents, male sterility of the parents is induced through a chemical hybridization agent, hybridization and backcross between the two types of materials are carried out, and the clubroot composite resistance germplasm is obtained. And a series of pure line germplasms with multiple resistance characters and phenotypic markers are bred by combining microspore culture, indoor resistance identification, molecular markers and phenotypic character assisted selection. And selecting corresponding resistance germplasm according to the target resistance gene required by each region, and inducing male sterility of the germplasm by using a hybridization agent to complete selective mating of the clubroot composite resistance hybrid variety. The method improves the utilization rate of the material and the efficiency and quality of artificial emasculation hybridization, can realize rapid polymerization of a plurality of resistance sites, and is suitable for popularization and application.

Description

Technical Field

[0001] The invention belongs to the technical field of germplasm resource creation, and relates to a method for creating a clubroot disease composite-resistant germplasm and an application thereof. Background Art

[0002] Clubroot is a worldwide soil-borne disease of cruciferous plants caused by the fungus Plasmodiophora brassicae Woronin, which infects the roots of over 100 species of crucifers, including rapeseed, Chinese cabbage, cabbage, radish, cauliflower, mustard, and shepherd's purse. It causes abnormal cell proliferation in the roots, leading to tumor formation, which in turn affects water and nutrient transport, causing above-ground leaf droop, slow plant growth, and even death. In recent years, clubroot has rapidly developed in my country, with the affected area expanding and spreading explosively, posing a serious threat to cruciferous crops. Controlling and managing this disease has become a critical issue for the healthy development of the rapeseed industry. Currently, clubroot control measures primarily include agricultural, biological, and chemical control, as well as the cultivation of clubroot-resistant varieties. Breeding and planting resistant varieties is the most cost-effective measure, while screening and developing clubroot-resistant germplasm is the foundation of disease-resistant breeding.

[0003] Studies have shown that European turnip (B.rapa L.ssp.rapifera) has strong resistance to clubroot. Traditional genetic studies have found that the clubroot resistance genes of cabbage (B.rapa, AA, 2n=20) are controlled by a few relatively independent major effect genes, and most of the resistance genes come from European forage turnip. Currently, eight clubroot resistance genes have been found in Chinese cabbage, including Crr1, Crr2, Crr3, Crr4, CRa, CRb, CRc and CRk. These CR genes all show dominant single gene inheritance; cabbage (B.oleracea, CC, 2n=18) has a quantitative resistance controlled by multiple genes. Its genetic basis for clubroot resistance is relatively complex, and at least 27 CR genes have been found. The QTLs for each gene are distinct; resistance to clubroot in Brassica napus (B. napus, AACC, 2n=38) is controlled by one or two independent dominant genes, which are present in a wide range of resistant accessions. The few key dominant genes or known major resistance loci for clubroot resistance all originate from the A genome of Brassica rapa (B. rapa). Brassica napus is the predominantly cultivated rapeseed variety in my country, but as an alloamphidiploid species with a relatively recent origin and a narrow genetic background, it lacks clubroot-resistant resources. Therefore, fully exploring and utilizing resistance genes in closely related species through hybridization and selection is an effective approach for developing clubroot-resistant germplasm and varieties. Research has shown that European turnip (ECD4) carries multiple clubroot-resistance genes. In Europe, Brassica napus was artificially synthesized by hybridizing turnip (ECD4) with resistant Brassica oleracea, resulting in the development of the clubroot-resistant Brassica napus variety Mendel. In my country, turnip (ECD4) was used as a donor parent and hybridized with the elite Brassica oleracea variety Huashuang 5. Through molecular marker-assisted selection, the conventional clubroot-resistant variety Huashuang 5R was developed, harboring the PbBa8.1 locus. Furthermore, the resistant Chinese cabbage variety Shinki was hybridized with a Brassica oleracea variety to develop the clubroot-resistant hybrid rapeseed variety Huayouza 62R, harboring the CRb locus. However, due to the diversity of clubroot races, pathogenicity varies between them, and dormant spores can survive in the soil for years, accumulating new mutations over time. Consequently, multiple species of clubroot can be isolated from the same diseased plot. Currently, most clubroot resistance loci exhibit vertical resistance rather than horizontal resistance, and single-gene resistance varieties exhibit strong race specificity. That is, a host plant harboring a specific resistance locus or gene may only be immune to a specific race and may not be resistant to other races. Long-term cultivation of a single resistant variety can lead to a loss of resistance.

[0004] Currently, the internationally recognized race identification systems for clubroot are the Williams system (with four identified hosts) and the ECD system (with 15 identified hosts). Domestic researchers have used the Williams system to identify samples of clubroot from different regions of my country and have found several major races: 1, 2, 3, 4, 7, 8, 11, and 13. Using the ECD system, samples collected have also revealed subspecies or variants of races 1, 2, 3, and 4. Race 4 has 10 subspecies, races 1 and 2 each have four subspecies, and races 3 and 8 each have three subspecies. Clubroot in most areas of my country is found in races 4 and 2. Resistance loci in resistant Brassica napus varieties are primarily derived from Brassica rapa (Brassica napus L.) and are generally PbBa8.1 or CRb loci, which are resistant to race 4. Studies have shown that different resistance loci have different resistance effects against different root-knot fungi, and there is a dosage effect between resistance genes and resistance. The aggregation of disease-resistance loci is beneficial for enhancing resistance levels. By simultaneously introducing multiple resistance loci into the same Brassica napus through polymer breeding, the disease resistance spectrum can be expanded and the variety's resistance to clubroot can be greatly improved. Therefore, identifying the physiological races of root-knot fungi from various regions, exploring new disease-resistance resources and identifying new disease-resistance loci, carrying out polymer breeding with multiple disease-resistance loci, cultivating high-quality rapeseed varieties that are simultaneously resistant to multiple physiological races of clubroot, and rationally and targetedly deploying them is the most effective way to prevent and control the occurrence and spread of the disease.

[0005] However, in the development of conventional clubroot-resistant rapeseed varieties, each generation requires field inoculation with clubroot pathogens to identify the genotype of intermediate materials. Furthermore, factors such as temperature, humidity, pathogen races, and the field distribution of the pathogen can affect the inoculation incidence rate, which in turn affects the correct selection and development of disease-resistant materials. While molecular marker-assisted selection can improve the efficiency of selecting resistant varieties by identifying genotypes in backcross populations, the currently developed clubroot-resistant gene markers are very limited or lack stability, leading to the loss of some non-marker resistance genes during the breeding process, impacting the aggregation of multiple resistance loci and the correct selection and development of disease-resistant materials. Other studies have shown that some morphological markers of Brassica napus (such as leaf color, flower color, and stem color) are intuitive and easily visible, suggesting that the use of dominant gene phenotypic markers for hybrid breeding has great potential.

[0006] In response to the problems existing in the prior art, the present invention provides a method for breeding a composite clubroot-resistant germplasm and its application, so as to achieve rapid aggregation of multiple resistance sites and provide a new method for breeding broad-spectrum and stable resistant germplasm and varieties. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for creating a clubroot compound-resistant germplasm and its application. The method is a method for breeding multi-resistant double-low cabbage rapeseed lines with compound resistance to clubroot by using indoor clubroot inoculation identification, phenotypic and molecular marker-assisted selection, and half-seed method analysis and selection. Using phenotypic trait materials controlled by a single gene and resistant materials identified by indoor inoculation of different clubroot races as parents, chemical hybridization agents are used to induce male sterility, and hybridization and backcrossing between the two types of materials are carried out. In combination with microspore culture, indoor resistance identification, molecular markers and phenotypic trait-assisted selection, a series of pure line germplasms with multiple resistance traits with phenotypic markers are bred. Then, the corresponding resistant germplasm is selected according to the target resistance genes required in each place, and the male sterility is induced by hybridization agents to complete the selection of clubroot compound-resistant hybrid varieties.

[0008] The specific technical solution is:

[0009] In one aspect, the present invention provides a method for creating a clubroot-resistant germplasm, the method comprising the following steps:

[0010] Step 1: According to the breeding goals, select single-gene-controlled phenotypic materials with outstanding target traits and good comprehensive traits (such as single-gene-controlled white flower materials 20ly295 and 20cly136; purple stem materials 19Cn34 and 20ly252) and clubroot-resistant materials (such as Huashuang 5R, 18hs7, 18hs8, 19hs8, 19Cn51, 19Cn154, etc.) in the material garden, and use multiple target strains to conduct indoor artificial inoculation identification of the selected resistant materials (according to the national agricultural industry standard: Technical Procedure for Identification of Rapeseed Clubroot Resistance NY / T3621-2020) and molecular marker selection (refer to the methods and procedures of Zhan Zongxiang et al. of the National Rapeseed Engineering and Technology Research Center of Huazhong Agricultural University and Huang Xinbiao et al. of the State Key Laboratory of Crop Improvement and Regulation in North China). 〔2〕 Select materials with phenotypic traits controlled by a homozygous single gene and materials with single dominant resistance to clubroot or materials with resistance controlled by relatively independent multiple dominant genes (known resistance marker + multiple unknown resistance markers).

[0011] Step 2: Planting the target trait material. Using a single dominant resistance material with a homozygous single unknown marker or a resistance material D controlled by relatively independent multiple dominant genes (known resistance marker + multiple unknown resistance markers) as the recipient parent, use a chemical hybridization agent to induce male sterility during the bud stage, and hybridize it with the phenotypic trait material B controlled by a homozygous single gene of the donor parent to obtain composite F1 rapeseed seeds containing both the phenotypic trait and the clubroot disease resistance locus;

[0012] Step 3: Plant the above-mentioned composite F1 rapeseed seeds, conduct resistance inoculation identification on individual plants with intermediate phenotypic traits, select target resistant individual plants, use chemical hybridization agents to induce male sterility in them during the bud stage, and backcross with a single dominant resistance material with a corresponding homozygous single unknown marker or a resistance material D (known resistance marker + multiple unknown resistance markers) controlled by relatively independent multiple dominant genes.

[0013] Step 4: Plant the above-mentioned backcross population, select individual plants with intermediate phenotypic traits between the phenotypic markers, and conduct resistance inoculation identification and molecular marker-assisted selection. Select target resistant individual plants, and for the backcross progeny of the single dominant resistant material with a homozygous single unknown marker, select individual plants with intermediate phenotypic traits for self-pollination; for the backcross progeny of the resistant material controlled by relatively independent multiple dominant genes (known resistance marker + multiple unknown resistance markers), select individual plants with intermediate phenotypic traits for molecular marker resistance identification, eliminate heterozygous resistant individual plants with known resistance markers based on the identification results, bag the homozygous resistant individual plants and self-pollinate to obtain self-pollinated seeds.

[0014] Step 5: Plant the aforementioned inbred lines and perform resistance inoculation testing based on the target resistance, eliminating inbred lines exhibiting resistance segregation. From the population of inbred lines that have not exhibited resistance segregation, select individual plants with homozygous phenotypic marker traits and bag them for self-pollination. Alternatively, after bud formation, remove 3.5-4.0 mm long flower buds from the main inflorescence and upper branch inflorescences of the plant, grind them in B5 medium, and then place them in NLN-13 medium supplemented with 0.5 mmol / L colchicine for doubling time for 36-48 hours. Then, switch to NLN-13 medium without colchicine for microspore induction to obtain a doubled haploid population. This creates single- or double-resistance target resistance material with homozygous phenotypic marker traits.

[0015] Step 6: Categorize and plant the resistant inbred lines with homozygous phenotypic markers according to the pathogen races they resist. Select resistant inbred lines with different genetic backgrounds, prominent target traits, and good overall traits (high resistance to multiple target clubroot races, high oil content, high yield, short stems, tolerance to dense planting, and resistance to lodging, etc.) based on the target resistance requirements. Induce male sterility in these inbred lines using chemical hybridization agents and cross them with normal, uninduced flowering plants. This will produce composite resistant germplasm or hybrid combinations with the target resistance.

[0016] The present invention provides the application of the method for creating clubroot composite-resistant germplasm in the rapeseed seed production process.

[0017] Compared with the prior art, the present invention combines a dominant morphological marker controlled by a single gene with intuitive and easily visible characteristics with molecular marker selection and traditional resistance identification technology to establish a breeding method for composite clubroot-resistant germplasm. Clubroot resistance selection can be performed based on the easy-to-observe and easy-to-select characteristics of morphological markers. This overcomes the problem that in the conventional rapeseed clubroot-resistant variety breeding process, each generation needs to use the field inoculation method of clubroot bacteria to identify the genotype of the intermediate material, and factors such as temperature, humidity, pathogen races, and field distribution of pathogens can affect the inoculation incidence rate, thereby affecting the correct selection of disease-resistant materials and the breeding process. This technology has higher breeding efficiency and clearer targets than conventional resistance selection methods. It also overcomes the problems that the specific markers of clubroot-resistant genes developed are very limited or have poor stability, resulting in the loss of some non-marker disease-resistant genes during the resistant variety breeding process, affecting the aggregation of multiple disease-resistant sites and the correct selection and breeding of disease-resistant materials. Secondly, this technology uses chemical hybridization technology with independent intellectual property rights for the selection of composite resistant varieties. It can arbitrarily select homozygous target resistant materials as hybrid parents, and use chemical hybridization agents to induce male sterility for hybridization selection, eliminating the sterile line conversion process, expanding the selection range of parents, and improving the utilization rate of materials. At the same time, the use of chemical hybridization agents to induce male sterility in rapeseed also improves the efficiency and quality of artificial emasculation hybridization, obtains sufficient resistant hybrid seeds, expands the selection group of individual plants, and increases the probability of selecting composite root root disease resistant germplasm, making it easier to achieve rapid aggregation of multiple resistance sites and complete the selection of broad-spectrum and stable resistant germplasm and varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the process for creating a clubroot-resistant germplasm of the present invention;

[0019] Figure 2 This is a schematic diagram of the process of Example 1 of the present invention;

[0020] Figure 3 This is a schematic diagram of the process of Example 3 of the present invention. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation plans.

[0022] The process of the method for creating a clubroot disease composite resistant germplasm of the present invention is as follows: Figure 1 shown.

[0023] Example 1: Creation of a composite germplasm resistant to white clubroot

[0024] The process of creating a compound resistant germplasm to white clubroot is as follows Figure 2 As shown, Figure 2 middle,

[0025] 1. Single gene dominant trait: Purple stem or white flower gene A;

[0026] 2. Recessive trait opposite to 1: Green stem or yellow flower gene a;

[0027] 3. Single gene dominant traits: clubroot resistance gene R;

[0028] 4. The recessive trait corresponding to 3: clubroot resistance gene r. The specific steps are as follows:

[0029] Step 1: Based on the breeding objectives, select a relatively independent resistant material D (carrying the PbBa8.1 resistance locus + multiple unknown resistance markers, and homozygous yellow flowers) from the material garden as the recipient parent. At the same time, select a pure white flower trait material B controlled by a single gene for high oil content, which has a prominent target trait and good overall characteristics, as the donor parent. During the rapeseed bud stage, use a chemical hybridization agent to induce male sterility in the resistant material, and hybridize it with the donor parent to obtain composite F1 rapeseed seeds containing both the white flower phenotype and the PbBa8.1 resistance locus + multiple unknown disease resistance loci.

[0030] Step 2: Plant the above-mentioned composite F1 rapeseed seeds, use different pathogen races for resistance inoculation identification, select individual plants with intermediate phenotypic traits (milky white flowers) and target resistance (eliminate yellow flower hybrid plants that are not normally hybridized), use chemical hybridization agents to induce male sterility during the bud stage, and backcross with the resistant material D (material carrying the PbBa8.1 resistance site + multiple unknown resistance markers) controlled by corresponding homozygous multiple dominant genes to obtain BC1F1 generation rapeseed seeds.

[0031] Step 3: Plant the BC1F1 generation of rapeseed seeds and eliminate yellow-flowered plants from the backcross population. For plants with an intermediate phenotypic trait (a creamy-white flower phenotype), select for prospects using molecular markers closely linked to PbBa8.1 and simultaneously identify resistance using the target pathogen race. Eliminate heterozygous resistant plants carrying the PbBa8.1 phenotype, and bag and self-pollinate homozygous resistant plants to obtain BC1F2 resistant rapeseed seeds carrying the PbBa8.1 resistance locus and an unknown resistance marker.

[0032] Step 4: Plant the above-mentioned BC1F2 generation rapeseed seeds. Based on the target resistance, select the pathogen races (e.g., races 2, 3, 4, and 7) to be inoculated and identified for resistance. Molecular markers closely linked to PbBa8.1 are used for prospect selection to eliminate inbred lines that exhibit resistance segregation. From the inbred line population that has not exhibited resistance segregation, select pure white or yellow flower plants with homozygous phenotypic marker traits, bag them, and self-pollinate them for multiple generations. Alternatively, after bud formation, remove 3.5-4.0 mm long flower buds from the main inflorescence and upper branch inflorescences of the plant, grind them in B5 medium, and then place them in NLN-13 medium supplemented with 0.5 mmol / L colchicine to double for 36-48 hours. Then, switch to NLN-13 medium without colchicine for microspore induction to obtain a doubled haploid population. That is, a pure line germplasm (LGKB) with pure white flower phenotypic marker traits + carrying PbBa8.1 resistance locus + composite resistance to target subspecies such as No. 2 or No. 3, 7, and 8 was created; and a new pure line germplasm (LGKH) with yellow flowers + carrying PbBa8.1 resistance locus + composite resistance to target subspecies such as No. 2 or No. 3, 7, and 8 was created.

[0033] Step 6: The resistant composite inbred line LGKB, which carries the pure white flower phenotypic marker, is planted according to the pathogen races it resists. Based on the target resistance, resistant inbred lines with distinct genetic backgrounds, prominent target traits, and good overall traits are selected. Chemical hybridization agents are used to induce male sterility in some plants of each inbred line, and these plants are then crossed with normal-flowering plants from other inbred lines. This results in the selection of a composite resistant material or hybrid LGKB1-x, which carries the pure white flower phenotypic marker and multiple target resistances. This has been identified as the white-flowered Brassica napus material 21LGKB31, which is highly resistant to clubroot races 2, 3, 4, 7, and 8.

[0034] Example 2: Creation of a purple stem and petiole clubroot-resistant germplasm

[0035] A method for creating a purple stem petiole clubroot-resistant germplasm comprises the following steps:

[0036] Step 1: Based on the breeding objectives, select a single dominant resistant material D with a homozygous unknown marker (not carrying the PbBa8.1 and CRb molecular markers, but highly resistant to clubroot races 3, 7, and 8, and homozygous for green stems and leaves) from the material garden as the recipient parent; at the same time, select a material B with a single gene for high oil content and a pure purple petiole trait that is prominent in the target trait and has good overall characteristics as the donor parent. In the rapeseed bud stage, use a chemical hybridization agent to induce male sterility in the above-mentioned resistant material, and hybridize it with the donor parent to obtain composite F1 rapeseed seeds that simultaneously contain the purple petiole phenotypic trait and highly resistant to clubroot races 3, 7, and 8.

[0037] Step 2: Plant the above-mentioned composite F1 rapeseed seeds, and use pathogen races 3 or 7 and 8 for resistance inoculation identification according to the local target pathogen races, select individual plants with intermediate phenotypic traits (light purple petioles) and target resistance (eliminate green stem and leaf hybrids that are not normally hybridized), use chemical hybridization agents to induce male sterility during the bud stage, and backcross with the corresponding homozygous single dominant resistance material D (does not carry PbBa8.1 and CRb molecular markers, but is highly resistant to clubroot physiological races 3, 7, and 8, and is homozygous for green stem and leaf materials) to obtain BC1F1 generation rapeseed seeds.

[0038] Step 3: Plant the aforementioned BC1F1 generation rapeseed seeds and eliminate plants with green stems and leaves from the backcross population. Plants with intermediate phenotypic traits (light purple petioles) are inoculated with the target pathogen race for resistance. Resistant plants are bagged and self-pollinated to obtain the target resistant rapeseed BC1F2 seeds.

[0039] Step 4: Plant the above-mentioned BC1F2 generation rapeseed seeds and select the pathogenic races (such as physiological races 3, 7, and 8) to be inoculated based on the target resistance for resistance inoculation identification. From the inbred line population that has not segregated for resistance, select single plants with pure purple petioles or pure green stems and leaves that have homozygous phenotypic marker traits, and bag them for continuous self-pollination. Alternatively, after bud formation, take 3.5-4.0 mm long flower buds from the main inflorescence and upper branch inflorescences of the plant, place them in B5 medium, grind them to extract microspores, and then place them in NLN-13 medium supplemented with 0.5 mmol / L colchicine to double for 36-48 hours. Then switch to NLN-13 medium without colchicine for microspore induction culture to obtain a doubled haploid population. That is, pure line germplasm (LGKZ) with pure purple petiole phenotypic marker traits + high resistance to target subspecies such as No. 3 or No. 7, No. 8 was created; and pure line germplasm (LGKG) with pure green stem and leaf phenotypic traits + high resistance to target subspecies such as No. 3 or No. 7, No. 8 was created.

[0040] Step 6: The resistant inbred lines LGKZ, bearing the purple petiole phenotypic marker, are planted according to the pathogen races they resist. Based on the target resistance, resistant inbred lines with different genetic backgrounds, prominent target traits, and good overall traits are selected. Chemical hybridization agents are used to induce male sterility in some plants of each inbred line, and these plants are then crossed with normal flowering plants of other inbred lines. This results in the selection of composite resistant materials or hybrids LGKZ1-x, which possess the purple petiole phenotypic marker trait and multiple target resistances. This has led to the development of a Brassica napus material LGKZx, bearing the purple petiole phenotypic marker trait and exhibiting high resistance to clubroot races 2, 3, 4, 7, and 8.

[0041] Example 3: Improvement of clubroot resistance in the new high-oil and lodging-resistant variety Qinyou 519

[0042] Methods for improving the clubroot resistance of the new high oil and lodging resistant variety Qinyou 519 Figure 3 As shown, the method specifically includes the following steps: Step 1: Using the Qinyou 519 female parent Q519A, which is not resistant to clubroot, as the recipient parent D, and based on the breeding goal, using the Brassica napus material LGKZ, which is homozygous for the purple petiole phenotype marker trait, carries the PbBa8.1 resistance locus, and is highly resistant to clubroot races 3 and 8, as the donor parent B. At the rapeseed bud stage, Q519A is induced to be male sterile using a chemical hybridization agent, and then hybridized with the donor parent to obtain composite F1 rapeseed seeds that simultaneously have the purple petiole phenotype trait and highly resistant to clubroot races 3, 4, and 8;

[0043] Step 2: Plant the above-mentioned composite F1 rapeseed seeds in an artificial climate chamber, and perform indoor resistance identification based on the local target pathogen race using molecular markers closely linked to PbBa8.1 and pathogen races 3, 4, and 8. Select individual plants with intermediate phenotypic traits (light purple petioles) and other phenotypic traits similar to Q519A(D) and target resistance (carrying the PbBa8.1 resistance locus + highly resistant to clubroot physiological races 3 and 8) (eliminate green stem and leaf hybrids that were not hybridized normally). Use chemical hybridization agents to induce male sterility in the bud stage, and backcross with Q519A(D) to obtain BC1F1 rapeseed seeds.

[0044] Step 3: Continue to plant the above-mentioned BC1F1 generation rapeseed seeds in an artificial climate chamber and eliminate individual plants with green stems and leaves in the backcross population. For individual plants with light purple petioles, use molecular markers closely linked to PbBa8.1 and pathogen races 3, 4, and 8 for indoor resistance identification, and compare with Q519A(D)'s own excellent traits such as long horns and lodging resistance. Select double-resistant individual plants with light purple petioles and other phenotypes similar to Q519A(D) as the female parent and backcross them with Q519A(D) for the second time. Repeat this backcross three times to obtain individual BC4F1 generation rapeseed seeds with target resistance (carrying the PbBa8.1 resistance locus + high resistance to clubroot races 3 and 8), light purple petioles, and other phenotypic traits identical to Q519A(D).

[0045] Step 4: Continue planting the aforementioned BC4F1 generation rapeseed seeds in an artificial climate chamber. During the flowering period, select individual plants from the BC4F1 generation population with light purple petioles and other phenotypic traits identical to those of Q519A(D) and bag them for self-pollination. Alternatively, after bud formation, remove 3.5-4.0 mm long flower buds from the main inflorescence and upper branch inflorescences of the plants, place them in B5 medium, grind them, and extract microspores. The buds are then placed in NLN-13 medium supplemented with 0.5 mmol / L colchicine for doubling time for 36-48 hours, and then switched to NLN-13 medium without colchicine for microspore induction. BC4F2 generation rapeseed self-pollination seeds and a doubled haploid population are obtained.

[0046] Step 5: Plant the above-mentioned BC4F2 generation rapeseed self-pollinated seeds and double haploid populations. Eliminate single plants with green stems and leaves and light purple petioles during the seedling stage, and select single plants with homozygous purple petiole phenotypic marker traits. Use molecular markers closely linked to PbBa8.1 and pathogenic races 3, 4, and 8 to perform indoor resistance identification on the selected single plants. Eliminate heterozygous single plants that do not carry the PbBa8.1 resistance locus and those that carry the PbBa8.1 resistance locus, as well as single plants that are not resistant to the target pathogenic race. Self-pollinate single plants that carry the PbBa8.1 homozygous resistance locus + highly resistant to clubroot races 3 and 8 + purple petiole phenotypic marker traits and whose other phenotypes are nearly the same as Q519A (D) in bags. Obtain BC4F3 generation rapeseed self-pollinated seeds.

[0047] Step 6: Plant the aforementioned BC4F3 inbred lines of rapeseed in separate rows in the field, along with the Qinyou 519 male parent, Q519C. These BC4F3 inbred lines are tested for resistance at the seedling stage using molecular markers closely linked to PbBa8.1 and target pathogen races 3, 4, and 8. Lines showing segregation of resistance to the target pathogen races are eliminated. Lines without segregation of resistance to the target pathogen races are retained. Individual plants carrying the homozygous PbBa8.1 resistance locus, high resistance to clubroot races 3 and 8, and the purple petiole phenotype are obtained, with other phenotypes nearly identical to those of Q519A(D). Some of these selected BC4F3 lines are bagged and self-pollinated. Other lines are subjected to chemical hybridization during the rapeseed bud stage to induce male sterility. These lines are then hybridized with the Qinyou 519 male parent, Q519C, at the flowering stage. A new improved variety Qinyou CR519 can be selected, which carries the PbBa8.1 resistance site + high resistance to clubroot physiological races 3 and 8 + light purple petiole phenotype.

[0048] The above description is only a preferred specific embodiment of the present invention, and the protection scope of the present invention is not limited thereto. Any simple change or equivalent replacement of the technical solution that can be obviously obtained by any technician familiar with this technical field within the technical scope disclosed in the present invention falls within the protection scope of the present invention.

Claims

1. A method for creating a clubroot-resistant germplasm, characterized in that: The following steps are involved: Step 1: selecting single-gene controlled phenotypic trait materials and clubroot disease-resistant materials in a material nursery according to the breeding goal, and performing indoor artificial inoculation identification and molecular marker selection on the selected resistant materials using multiple target strains to obtain target trait materials, wherein the target trait materials include identified and selected homozygous single-gene controlled phenotypic trait materials and clubroot disease single dominant resistance materials or resistance materials controlled by relatively independent multiple dominant genes; Step 2: Planting the target trait material described in step 1, using a single dominant resistance material with a homozygous single unknown marker or a resistance material controlled by relatively independent multiple dominant genes D as the recipient parent, inducing male sterility in the material using a chemical hybridization agent at the bud stage, and hybridizing it with the phenotypic trait material B controlled by a homozygous single gene of the donor parent to obtain composite F1 rapeseed seeds containing both the phenotypic trait and the clubroot disease resistance locus; Step 3: Planting the composite F1 rapeseed seeds, performing resistance inoculation identification on individual plants with intermediate phenotypic traits, selecting target resistant individual plants, inducing male sterility in them using chemical hybridization agents at the bud stage, and backcrossing them with single dominant resistant materials with corresponding homozygous single unknown markers or resistant materials D controlled by relatively independent multiple dominant genes to obtain a backcross population; Step 4: Planting the backcross population described in step 3, selecting individual plants with intermediate phenotypic traits of phenotypic markers, performing resistance inoculation identification and molecular marker-assisted selection, selecting target resistant individual plants, and self-pollinating the backcross progeny of the single dominant resistant material with a homozygous single unknown marker, selecting individual plants with intermediate phenotypic traits; for the backcross progeny of the resistant material controlled by relatively independent multiple dominant genes, selecting individual plants with intermediate phenotypic traits for molecular marker resistance identification, eliminating heterozygous resistant individual plants with known resistance markers based on the identification results, bagging the homozygous resistant individual plants and self-pollinating to obtain inbred lines; Step 5: Planting the inbred lines described in step 4, performing resistance inoculation identification on them according to the target resistance, eliminating inbred lines that show resistance segregation, selecting individual plants with homozygous phenotypic marker traits from the inbred line population that has not shown resistance segregation, bagging and self-pollinating, or taking flower buds with a length of 3.5-4.0 mm on the main inflorescence and upper branch inflorescence of the plant after bud formation, placing them in B5 medium, grinding them to extract microspores, and then placing them in NLN-13 medium supplemented with 0.5 mmol / L colchicine for doubling for 36-48 hours, and then switching to NLN-13 medium without colchicine for microspore induction culture to obtain a double haploid population, thereby creating an inbred line with single or double resistance target resistance material and homozygous phenotypic marker traits; Step 6: The above-mentioned inbred lines of resistant materials with homozygous phenotypic marker traits are classified and planted according to the pathogen subspecies they resist. According to the target resistance requirements, resistant inbred lines with different genetic backgrounds and outstanding target traits are selected, and chemical hybridization agents are used to induce male sterility in some plants of each inbred line, which are then hybridized with normal flower plants that have not been induced to select composite resistant germplasm or hybrid combinations with target resistance.

2. The method for creating a clubroot-resistant germplasm according to claim 1, characterized in that: In step 1, materials with phenotypic traits controlled by a homozygous single gene are selected, and multiple target strains are used to perform indoor artificial inoculation identification and molecular marker selection on resistant materials, and single dominant resistance materials for clubroot or resistance materials controlled by multiple relatively independent dominant genes are selected.

3. The method for creating a clubroot-resistant germplasm according to claim 2, characterized in that: A single dominant resistant material with a homozygous single unknown marker or a resistant material D controlled by relatively independent multiple dominant genes is used as the recipient parent. Chemical hybridization agents are used to induce male sterility during the bud stage, and the material is hybridized with the phenotypic trait material B controlled by a homozygous single gene of the donor parent to obtain composite F1 rapeseed seeds containing both phenotypic traits and clubroot disease resistance sites.

4. The method for creating a clubroot-resistant germplasm according to claim 3, characterized in that: Planting the F1 rapeseed seeds described in claim 2, and using the indoor artificial inoculation identification method for clubroot disease described in claim 1 to perform resistance inoculation identification on individual plants with intermediate phenotypic traits, selecting target resistant individual plants, inducing male sterility in them using chemical hybridization agents at the bud stage, and backcrossing them with a single dominant resistance material having a corresponding homozygous single unknown marker or a resistance material D controlled by relatively independent multiple dominant genes.

5. The method for creating a clubroot-resistant germplasm according to claim 4, characterized in that: Plant the backcross population obtained in claim 3, select individual plants with intermediate phenotypic traits of phenotypic markers, and use the clubroot disease resistance identification method described in claim 1 to perform resistance inoculation identification and molecular marker-assisted selection on them, eliminate heterozygous resistant individual plants with known resistance markers based on the identification results, and bag and self-pollinate the homozygous resistant individual plants.

6. The method for creating a clubroot-resistant germplasm according to claim 5, characterized in that: The inbred line obtained in claim 4 is planted, and the clubroot resistance identification method according to claim 1 is used to perform resistance inoculation identification on it, and the inbred lines with resistance segregation are eliminated. Single plants with homozygous phenotypic marker traits are selected for bagging and self-pollination, or flower buds with a length of 3.5-4.0 mm on the main inflorescence and upper branch inflorescence of the plant are taken after budding, placed in B5 medium, and ground to extract microspores. Subsequently, the buds are placed in NLN-13 medium supplemented with 0.5 mmol / L colchicine for doubling for 36-48 hours, and then switched to NLN-13 medium without colchicine for microspore induction culture to obtain a double haploid population, and create a single-resistance or double-resistance target resistance material with homozygous phenotypic marker traits.

7. Use of the method for creating a clubroot-resistant composite germplasm according to any one of claims 1 to 6 in rapeseed seed production.

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