Breeding method of clubroot-resistant brassica napus

Abstract

The invention belongs to the technical field of breeding, and particularly relates to a breeding method of clubroot-resistant brassica napus. The method comprises the following steps: by taking a clubroot-resistant brassica napus strain as a material, inducing to obtain a double haplobiont population, identifying and selecting plants with good fertility, up-to-standard seed quality and high clubroot resistance, hybridizing with a cytoplasmic genic male sterile line of the brassica napus to obtain an F1 generation, planting the seeds of the F1 generation, and identifying to obtain the clubroot-resistant brassica napus cytoplasmic genic male sterile line of the brassica napus. The method comprises the following steps: selecting a plant which is a restoring line of a cabbage type rape cytoplasmic genic male sterile line and a clubroot-resistant line and has F1-generation seed quality reaching the standard as a candidate male parent, hybridizing the candidate male parent with the cabbage type rape cytoplasmic genic male sterile line to obtain F1-generation seeds, identifying the F1-generation seeds, and identifying the F1-generation seeds to obtain the cabbage type rape cytoplasmic genic male sterile line. And selecting a candidate male parent and a candidate female parent which are high in clubroot resistance and excellent in comprehensive character performance to hybridize and combine, namely the clubroot-resistant brassica napus variety.

Description

Technical Field

[0001] The invention belongs to the technical field of breeding, and in particular relates to a breeding method for clubroot-resistant Brassica napus. Background Art

[0002] Rapeseed is the second largest oil crop in the world and the fifth largest crop after rice, wheat, corn and soybean. As a major oil crop, rapeseed not only provides safe, healthy and nutritious edible oil and industrial raw materials for humans, but also provides high-quality protein-rich feed for herbivores.

[0003] Rapeseed is susceptible to various diseases and infections due to its long growth period. Common diseases of rapeseed include sclerotinia, downy mildew, powdery mildew and clubroot. Clubroot is a common soil-borne disease of Brassica plants. When the plant is infected, the roots swell, gradually destroying the root tissue, and the inability to absorb water causes the aboveground part to wither and die. In recent years, with the mechanization of crop operations and cross-provincial and cross-regional operations, extremely convenient conditions have been created for the spread of clubroot bacteria. Clubroot spreads quickly and is severe, which seriously restricts the normal production of rapeseed.

[0004] Brassica napus is one of the three major cultivated types of rapeseed. It is widely cultivated for its advantages such as high oil content, high yield, resistance to barrenness, stress resistance, stable yield and great potential for yield increase. Due to the lack of effective antigens, the difficulty of breeding rapeseed resistant to clubroot has increased. At present, the effective method for breeding rapeseed resistant to clubroot is to first introduce the anti-clubroot antigens in turnip into cabbage, and then gradually introduce them from cabbage into Brassica napus. Although this method is feasible, with the years of planting of clubroot-resistant rapeseed varieties, the differentiation rate of its pathogens is too fast, resulting in a large number of physiological subspecies. Varieties with disease-resistant genes with a single or a small number of physiological subspecies have weak disease resistance and short disease resistance, resulting in a large-scale outbreak of rapeseed clubroot, which seriously affected the yield and quality of rapeseed and dampened the enthusiasm of farmers for production. They would rather abandon their fields than plant rapeseed. Varieties with multiple disease-resistant genes can resist pathogens of multiple physiological subspecies, and have stronger and more lasting disease resistance.

[0005] In existing breeding technology, traditional rapeseed varieties are not resistant to clubroot. When rapeseed plants are infected by clubroot bacteria, the underground root tissue is gradually destroyed by the bacteria, which manifests as swollen roots that resemble ginger pieces. During the seedling stage, the rapeseed plants have a low demand for water, so they grow slowly and are weak. After the bud stage, the rapeseed plants grow vigorously and have a high demand for water. The aboveground parts often wilt around noon due to insufficient water supply to the roots and strong transpiration of the leaves, resulting in slow growth, delayed flowering, and plant death in severe cases. The yield drops sharply or even fails. In traditional rapeseed breeding, the pathogen responsible for resistance to clubroot comes from turnip. By introducing the pathogen, some poor quality traits such as erucic acid, glucosinolates and other unfavorable quality traits are also introduced, which also increases the difficulty of improving the quality of rapeseed in the later stage. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a breeding method for clubroot-resistant Brassica napus.

[0007] The present invention adopts the following technical solution:

[0008] A method for breeding clubroot-resistant Brassica napus, comprising the following steps:

[0009] S1. Selecting clubroot-resistant Brassica napus as materials for cultivating double haploid plants, inducing a double haploid plant population, and selecting double haploid plants that are naturally fully mature, have good fertility, meet seed quality standards, and are highly resistant to clubroot;

[0010] S2, hybridizing the double haploid plant of S1 as the male parent and the cytoplasmic nuclear sterile male sterile line of Brassica napus as the female parent to obtain the second F1 generation, and selecting the male parent of the double haploid plant which is both a restorer line of the cytoplasmic nuclear sterile male sterile line of Brassica napus and a clubroot-resistant line and whose second F1-generation seed quality meets the standard as the candidate male parent for cultivating clubroot-resistant Brassica napus;

[0011] S3, using the cytoplasmic nuclear sterile male sterile line of Brassica napus as a candidate female parent for cultivating Brassica napus resistant to clubroot;

[0012] S4. Hybridize the candidate male parent and the candidate female parent, harvest the seeds of the third F1 generation, and conduct comprehensive identification of clubroot disease resistance and agronomic traits, and select the hybrid combination of the candidate male parent and the candidate female parent with strong clubroot disease resistance and excellent comprehensive trait performance, which is the clubroot disease-resistant Brassica napus variety.

[0013] The present invention uses a cytoplasmic nuclear sterile male sterile line of cabbage-type rapeseed as a female parent, and uses a double haploid plant that is both a restorer line of the cytoplasmic nuclear sterile male sterile line of cabbage-type rapeseed and a clubroot disease-resistant line and whose second F1 generation seed quality meets the standard as a male parent, and obtains a cabbage-type rapeseed variety resistant to clubroot disease by hybridization of the two. The method of the present invention does not require the introduction of turnip genes, and the obtained cabbage-type rapeseed variety resistant to clubroot disease has no introduction of adverse quality traits such as erucic acid and glucosinolate, and has excellent comprehensive performance in clubroot disease resistance, lodging resistance, initial flowering period, final flowering period, maturity period, 1,000-grain weight, seed oil content and yield.

[0014] Furthermore, in S1, the selection of clubroot-resistant rapeseed lines is based on the clubroot disease index DI as a screening index:

[0015] Germplasm with disease index DI < 5 is identified as highly resistant to clubroot, 5≤DI < 15 is resistant to clubroot, 15≤DI < 30 is tolerant to clubroot, 30≤DI < 50 is susceptible to clubroot, and ≥50 is susceptible to clubroot.

[0016] Furthermore, in S1, the selection of clubroot-resistant rapeseed lines specifically comprises the following steps:

[0017] Step 1: Select pure line materials with different genetic backgrounds and specific traits from the Brassica napus germplasm resource bank as the first germplasm;

[0018] Step 2: Identify the first germplasm for its resistance to clubroot disease, and use the germplasm resources with a clubroot disease index of less than 30 as the second germplasm;

[0019] Step 3: The second germplasm is identified for resistance to clubroot, and the second germplasm with a clubroot disease index of less than 15, excellent comprehensive agronomic traits and up-to-standard seed quality in six consecutive rounds of identification is selected as the third germplasm;

[0020] Step 4: Using the third germplasm as a parent, artificially hybridizing different germplasms to obtain the first F1 generation of different hybrid combinations;

[0021] Step 5: The first F1 generation is identified for its resistance to clubroot disease, and the parent combination corresponding to the first F1 generation with a clubroot disease index <5 is used as material for cultivating double haploid plants.

[0022] Furthermore, the comprehensive excellent agronomic traits refer to compact plant type, moderate plant height, early maturity, and good fruit set;

[0023] Furthermore, the seed quality meeting the standard means that the seed oil content is greater than 40%, the seed glucosinolate content is less than 30 μmol / g cake and the seed erucic acid content is less than 3%.

[0024] Furthermore, in S1, inducing a double haploid plant population specifically includes:

[0025] At the bolting and budding stage, the flower buds at the mononuclear marginal stage of the material for cultivating double haploid plants are taken for microspore culture. During the process of embryo formation, colchicine is added to double the chromosomes. After the embryos appear, they are differentiated and induced into seedlings to obtain a double haploid plant population.

[0026] Furthermore, S5 also includes the identification of resistance to Sclerotinia sclerotinia and the identification criteria for the incidence of Sclerotinia sclerotinia:

[0027] Grade 0: no symptoms;

[0028] Level 1: Less than 1 / 3 of the branches are diseased or the main stem has small lesions, and less than 1 / 4 of the siliques on the whole plant are affected;

[0029] Level 2: 1 / 3-2 / 3 of the branches or the middle and upper part of the main stem are affected, and 1 / 4-2 / 4 of the siliques on the whole plant are affected;

[0030] Level 3: more than 2 / 3 of the branches or the middle and lower part of the main stem are diseased, and 2 / 4-3 / 4 of the siliques on the whole plant are affected;

[0031] Level 4: All or most of the branches are infected or there are lesions around the base of the main stem, and up to 3 / 4 of the siliques on the whole plant are infected.

[0032] Furthermore, in S5, the sclerotinia disease incidence level of the clubroot-resistant Brassica napus variety is less than level 1.

[0033] The present invention has the following beneficial effects:

[0034] The Brassica napus variety cultivated by the breeding method of the present invention not only obtains the characteristic of high resistance to clubroot, but also obtains the ability of resistance to sclerotinia sclerotinia.

[0035] The present invention obtains a cabbage-type rapeseed germplasm material with high resistance to clubroot, sclerotinia and lodging, which provides important technical support and alternative resources for high-quality rapeseed research and new variety breeding.

[0036] The hybrid combinations RU12×Shaan 3A, RU38×Shaan 3A, RU122×Shaan 3A and RU201×Shaan 3A obtained by the breeding method of the present invention are early-maturing, have good firmness and high yield, and are suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 These are rapeseed clubroot susceptible germplasm (left) and clubroot resistant germplasm (right).

[0038] Figure 2 It is the classification standard for clubroot disease grade identification.

[0039] Figure 3 This is a picture of the rapeseed microspore culture experiment.

[0040] Figure 4 This is a picture of rapeseed DH strain test tube seedlings.

[0041] Figure 5 This is a picture of field bagged self-pollination of test tube seedlings.

[0042] Figure 6 These are pictures of the root systems of rapeseed varieties susceptible to clubroot (left) and rapeseed varieties resistant to clubroot (right).

[0043] Figure 7 These are the root systems of four rapeseed varieties resistant to clubroot.

[0044] Figure 8This is the growth condition of rapeseed resistant to clubroot when it matures. DETAILED DESCRIPTION

[0045] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0046] The genetic resources of the following examples of the present invention are from the "Rapeseed Germplasm Resource Bank of the Department of Agronomy of Ankang College". 348 pure line materials with different genetic backgrounds and specific traits are disclosed in the following documents:

[0047] Zhao Weiguo, Tana, Wang Hao. QTL positioning and candidate gene analysis of glucosinolate content in seeds of Brassica napus[J]. Journal of Northwest Botany, 2024, 44(8): 1261-1272.

[0048] The cytoplasmic nuclear sterile male sterile line of Brassica napus “Shaan 3A” is published in the following documents:

[0049] Li Dianrong, Tian Jianhua, Chen Wenjie, Ren Junrong, Mu Jianxin, Zhang Xin, Wang Hao. Breeding, hybrid production and purity detection of high-quality rapeseed variety Qinyou 7[J]. Journal of Northwest Agriculture, 2010, 19(3): 87-92.

[0050] Example 1

[0051] A method for breeding clubroot-resistant Brassica napus, comprising the following steps:

[0052] 1. Collection, evaluation and identification of basic germplasm, screening out germplasm resources with clubroot tolerance and resistance in soil environments where clubroot outbreaks occur year-round; specifically including:

[0053] (1) A total of 348 pure line materials with different genetic backgrounds and specific traits were selected from the Brassica napus germplasm resource bank as germplasm resources.

[0054] (2) 348 accessions of Brassica napus germplasm resources were planted in an experimental field where clubroot had broken out for many years (alpine vegetable-cabbage planting fields in Shanghe Village, Taibai County, Shaanxi Province). At the same time, favorable conditions were created according to the environmental requirements for clubroot (temperature, soil moisture, and weakly acidic environment) to promote the severity of rapeseed clubroot. The temperature was adjusted according to the environment during the sowing period. If the sowing was carried out on April 20, rapeseed clubroot would be severely affected in late June and early July. In July, due to the high altitude of Taibai County, the temperature could not meet the requirements for the occurrence of clubroot, and clubroot almost did not occur. Soil moisture can be solved through irrigation measures. Clubroot requires a moist soil environment. In addition, clubroot requires an acidic soil environment. Because Taibai County is located at the watershed between the Yangtze River Basin and the Yellow River Basin, some fields are weakly alkaline and some are weakly acidic. Generally, a certain amount of acidic fertilizer ammonium phosphate is applied at 20 kg per mu to adjust the soil acidity and alkalinity.

[0055] (2) When the seedlings were 60 days old, 5 plants were selected from each row of each rapeseed germplasm, and a total of 10 plants in 2 rows were dug out with their roots to identify whether the plant lines were diseased ( Figure 1 ) ; Statistics on the incidence of rapeseed clubroot and disease grade identification.

[0056] Incidence rate (%) = number of diseased plants / total number of surveyed plants × 100%.

[0057] Rapeseed clubroot disease level identification standard: Level 0, no tumors on the roots; Level 1, a small number of small tumors on the fibrous roots; Level 3, tumors on the lateral roots; Level 5, large tumors on the lateral roots and small tumors on the taproot tips; Level 7, all lateral taproots are swollen, but the plant grows normally; Level 9, most of the swollen roots begin to crack and rot, and the plant wilts or has died ( Figure 2 ).

[0058] (3) When the seedlings were 60 days old, the incidence of clubroot in different germplasm resources was counted, and the severity and grade of clubroot in each germplasm were investigated. Diseased plants were graded and counted according to the disease grade identification standard for rapeseed clubroot, and the disease index (DI) of each germplasm was calculated.

[0059] The resistance to clubroot is divided into different levels according to the disease index of different germplasms. Germplasm with a disease index DI < 5 is identified as highly resistant to clubroot, 5≤DI < 15 is resistant to clubroot, 15≤DI < 30 is resistant to clubroot, 30≤DI < 50 is susceptible to clubroot, and ≥50 is susceptible to clubroot, the same below.

[0060] According to the disease index classification, disease-resistant germplasm and germplasm resources with a disease index lower than that of disease-resistant germplasm are regarded as target germplasm resources resistant to severe diseases.

[0061] 2. Select germplasm resources with compact plant type, moderate plant height, early maturity, good fruit set, high seed oil content and low glucosinolate and erucic acid from the germplasm resources of S1 with clubroot resistance and disease resistance, and mix and hybridize them to obtain the first F1 generation of hybrids of different parents, and select high-quality clubroot-resistant strains from them, including:

[0062] (1) The target germplasm resources were subjected to six rounds of field clubroot resistance tests in the clubroot identification test field for three consecutive years, and 21 germplasm resources with a disease index DI < 15 in the six rounds were selected as candidate germplasms for breeding clubroot-resistant varieties (QT26, QT32, QT33, QT52, QT53, QT54, QT65, QT66, QT98, QT99, QT126, QT138, QT162, QT171, QT172, QT174, QT189, QT206, QT213, QT254, QT255);

[0063] (2) From 21 germplasm resources, we screened 7 lines with compact plant type (branch angle less than 30°), moderate plant height (below 170 cm), and early maturity (mature before June). At the same time, we combined them with good fruit set (able to bloom, pollinate and produce plump grains), seed oil content >40%, and seed double low quality (glucosinolate content <30 μmol / g cake, erucic acid content <3%), namely QT32, QT52, QT53, QT98, QT174, QT206, and QT213.

[0064] (3) The above 7 germplasms were used as parents, and artificial hybridization between different germplasms was performed to obtain 42 combinations of F1 generations from hybridization of different parents (pairwise hybridization, including forward and reverse crosses).

[0065] (4) The 42 F1 combinations were sown in the clubroot disease identification test field and identified for clubroot disease at the seedling stage. The combinations with a disease index DI < 5 were selected as clubroot disease resistant strains. A total of 4 clubroot disease resistant strains were screened, namely QT32×QT98, QT32×QT206, QT53×QT98, and QT174×QT98.

[0066] 3. Using the clubroot-resistant strains of S2 as materials, a double haploid (DH) plant population was obtained by inducing the double single embryo induction technology of Brassica napus, and the seeds of naturally fully mature and fertile double haploid plants were tested for quality, and seeds of the quality-qualified strains were retained, and their clubroot resistance was identified, and the clubroot-resistant strains of the DH population cultured from the two hybrid combinations of QT32×QT98 and QT53×QT98 in S2 were retained; specifically:

[0067] (1) In the bolting and budding stage, two strains with good comprehensive traits in the seedling stage were selected from each combination. The flower buds (flower bud diameter 1-2 mm) at the mononuclear edge stage were taken for microspore culture ( Figure 3 ), put the flower buds into steel baskets at the appropriate time, sterilize with 70% alcohol for 30 seconds, 0.1% mercuric chloride for 8-10 minutes, wash three times with sterile water, crush in B5-13 culture medium, filter with 300 mesh nylon mesh, wash by centrifugation, suspend and precipitate in NLN culture medium, treat 3 dishes for each material, culture at 32-33℃ for 36-48h, replace with new culture medium by centrifugation, transfer to 23-25℃ dark culture for more than two weeks, transfer to shaking table culture after embryoids are visible to the naked eye, add colchicine solution with a mass concentration of 0.1% to double chromosomes during embryo formation, transfer to B5-13 solid culture medium for differentiation and induction into seedlings after embryos are visible (for details, refer to Zhao Xiaoping's Optimization Research on High-efficiency Induction Technology System of Double Haploid of Cabbage Rape), and obtain double haploid (DH) plants ( Figure 4 ), test tube seedlings cultured from microspores, self-pollination in bags to ensure homozygosity of each material ( Figure 5 ).

[0068] (2) The doubled haploid plants were planted in a test field without root knot fungus (the doubled haploid plants have relatively weak growth and poor disease resistance), and fertility was identified during the flowering period to select strains with good fertility.

[0069] (3) The seeds of naturally fully mature and good fertility lines were tested for quality using near-infrared reflectance spectroscopy (NIRS) and nuclear magnetic resonance (NMR), and the seeds of the lines that met the quality standards (oil content > 40%, glucosinolate content < 30 μmol / g cake, erucic acid content < 3%) were retained.

[0070] (4) For the strains that met the quality requirements, the clubroot disease resistance was identified using the same two methods (clubroot disease identification test field and laboratory artificial inoculation of clubroot fungi). Thirteen clubroot disease-resistant strains (RU05, RU12, RU25, RU38, RU58, RU73, RU99, RU122, RU135, RU178, RU201, RU224, and RU245) with a disease index DI less than 5 using both methods were retained.

[0071] Among them, RU05, RU12, RU25, RU38, RU58, RU73, RU99, RU122, and RU135 were obtained by the hybrid combination culture of QT32×QT98.

[0072] The strains obtained by hybridization of QT53×QT98 are RU178, RU201, RU224 and RU245.

[0073] 4. During the flowering period, the 13 clubroot-resistant strains in (4) of S3 were used as male parents and hybridized with Shaan 3A to obtain the F1 generation. The seeds of the F1 generation were planted in a test field where clubroot disease occurs all year round. The hybrid combinations RU12×Shaan 3A, RU38×Shaan 3A, RU122×Shaan 3A, and RU201×Shaan 3A with excellent comprehensive agronomic traits and high resistance to clubroot were selected. The male parents RU12, RU38, RU122, and RU201 corresponding to the four excellent combinations were selected as candidate male parents for breeding clubroot-resistant Brassica napus.

[0074] Specifically include:

[0075] (1) Some F1 seeds of different hybrid combinations were cultured in an artificial climate chamber and vernalized to make them bloom quickly. The male parents of the hybrid combinations RU12, RU25, RU38, RU58, RU99, RU122, RU135, RU178, RU201, and RU224 with excellent comprehensive traits of normal fertility, yield, oil content, flowering period, maturity period, quality, and lodging resistance were selected as the restorer lines of the cabbage-type cytoplasmic nuclear sterile male sterile line Shaanxi 3A.

[0076] (2) Another portion of the F1 seeds were sown in the clubroot disease identification test field to identify the disease status of different strains. Six strains of highly resistant clubroot disease with a disease index DI less than 5, namely RU05, RU12, RU38, RU122, RU135, and RU201, were selected as candidate male parents for breeding highly resistant clubroot disease varieties ( Figure 6 ).

[0077] (3) Select a strain that is both a restorer of Shaan 3A and highly resistant to clubroot ( Figure 7 ), and the male parent lines corresponding to the combination of F1 generation seed quality-compliant lines (oil content > 40%, glucosinolate content < 30 μmol / g cake, erucic acid content < 3%) were selected as candidate male parents for breeding clubroot-resistant elite varieties (RU38, RU122, RU135, RU201). The seed quality test data are shown in Table 1.

[0078] Table 1 Seed quality test data

[0079] serial number RU38 RU122 RU135 RU201 Oil content% 41.29 42.96 41.26 42.03 Glucosinolate μmol / g 25.26 14.72 14.14 17.46 Erucic acid% 1.61 1.43 1.49 1.86 protein% 31.44 29.19 28.64 28.1 Palmitic acid% 4.83 3.86 4.43 3.92 Stearic acid% 2.57 1.84 1.96 2.31 Oleic acid% 64.59 62.73 63.91 65.3 Linoleic acid% 19.9 16.07 19.09 14.64 Linolenic acid% 8.42 10.49 9.58 8.72 Arachidonic acid% 0 0.38 0.54 0.05 Palmitoleic acid% 0.2 0.2 0.2 0.2 Arachidic acid % 0.28 0.32 0.33 0.52 Arachidadienoic acid% 0 0.02 0.04 0.04 % Docosanoic acid 0 0.06 0.02 0.07

[0080] 5. Use Shaanxi 3A as a candidate maternal line for breeding high-quality varieties resistant to clubroot disease.

[0081] 6. Hybridize the candidate male parent line of S4 with the candidate female parent line of S5 to obtain F1 generation seeds, and conduct comprehensive identification of clubroot disease resistance and agronomic traits on them again, and select hybrid combinations with clubroot disease resistance and excellent comprehensive agronomic traits, including:

[0082] Seed production was carried out in the net house to obtain F1 seeds of regional trials, and their field test maturity (in Ankang area, the maturity should be harvested before May 10), lodging resistance (rapeseed stems are upright, and the main inflorescence can be semi-lodged), quality (oil content>40%, glucosinolate content<30μmol / g cake, erucic acid content<3%) and disease resistance (sclerotinia resistance (sclerotinia disease incidence level<1), high resistance to clubroot, that is, disease index DI<5) were identified, and the lines with high resistance to clubroot and excellent comprehensive traits were selected ( Figure 8 );

[0083] The F1 generations of the hybrid combinations RU12×Shaan3A, RU38×Shaan3A, RU122×Shaan3A, and RU201×Shaan3A, which were configured using clubroot-resistant rapeseed lines, all showed resistance to clubroot (Table 2).

[0084] Table 2 Disease index of clubroot in F1 generation of different combinations of rapeseed

[0085]

[0086] Sclerotinia identification standards:

[0087] Grade 0: no symptoms;

[0088] Level 1: Less than 1 / 3 of the branches are diseased or the main stem has small lesions, and less than 1 / 4 of the siliques on the whole plant are affected;

[0089] Level 2: 1 / 3-2 / 3 of the branches or the middle and upper part of the main stem are affected, and 1 / 4-2 / 4 of the siliques on the whole plant are affected;

[0090] Level 3: more than 2 / 3 of the branches or the middle and lower part of the main stem are diseased, and 2 / 4-3 / 4 of the siliques on the whole plant are affected;

[0091] Level 4: All or most of the branches are infected or there are lesions around the base of the main stem, and up to 3 / 4 of the siliques on the whole plant are infected.

[0092] The hybrid combinations RU12×Shaan 3A, RU38×Shaan 3A, RU122×Shaan 3A, and RU201×Shaan 3A were configured using rapeseed clubroot-resistant lines. The F1 generation of RU122×Shaan 3A was resistant to sclerotinia sclerotinia, the F1 generation of RU12×Shaan 3A and RU201×Shaan 3A was resistant to sclerotinia sclerotinia, but the F1 generation of RU38×Shaan 3A was susceptible to sclerotinia sclerotinia (Table 2).

[0093] Table 3 Identification of Sclerotinia sclerotiorum in F1 generation of different combinations of rapeseed

[0094]

[0095] 7. The hybrid combination strains with excellent comprehensive traits in S6 were used as excellent strains of rapeseed resistant to clubroot for field trials. The yield of the plots (20 m 2)(Table 4), lodging resistance, initial flowering period, final flowering period, maturity period, 1000-grain weight and seed oil content (Table 5) and comprehensive performance of variety comparison test, finally obtained rapeseed clubroot resistant variety (hybrid combination RU122×Shaan 3A). In addition, the male parent of rapeseed Qinyou 3R is the clubroot resistant line RU122, which also shows the characteristics of clubroot resistance, but the clubroot resistance and yield are lower than RU122×Shaan 3A.

[0096] Table 4 Yield investigation of F1 plots of different rapeseed combinations

[0097]

[0098] Table 5 Investigation of other traits of F1 generation of different combinations of rapeseed

[0099]

[0100] Table 6 Comparison of yield of rapeseed resistant clubroot combination RU122×Shaan 3A with other varieties

[0101]

[0102]

[0103] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0104] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for breeding clubroot-resistant Brassica napus, characterized in that: The following steps are involved: S1. Selecting clubroot-resistant Brassica napus as materials for cultivating double haploid plants, inducing a double haploid plant population, and selecting double haploid plants that are naturally fully mature, have good fertility, meet seed quality standards, and are highly resistant to clubroot; S2, hybridizing the double haploid plant of S1 as the male parent and the cytoplasmic nuclear sterile male sterile line of Brassica napus as the female parent to obtain the second F1 generation, and selecting the male parent of the double haploid plant which is both a restorer line of the cytoplasmic nuclear sterile male sterile line of Brassica napus and a clubroot-resistant line and whose second F1-generation seed quality meets the standard as the candidate male parent for cultivating clubroot-resistant Brassica napus; S3, using the cytoplasmic nuclear sterile male sterile line of Brassica napus as a candidate female parent for cultivating Brassica napus resistant to clubroot; S4. Hybridize the candidate male parent and the candidate female parent, harvest the seeds of the third F1 generation, and conduct comprehensive identification of clubroot disease resistance and agronomic traits, and select the hybrid combination of the candidate male parent and the candidate female parent with strong clubroot disease resistance and excellent comprehensive trait performance, which is the clubroot disease-resistant Brassica napus variety.

2. The method for breeding clubroot-resistant Brassica napus according to claim 1, characterized in that: In S1, the clubroot disease index DI was used as the screening index for selecting clubroot-resistant rapeseed lines: Germplasm with disease index DI < 5 is identified as highly resistant to clubroot, 5≤DI < 15 is resistant to clubroot, 15≤DI < 30 is tolerant to clubroot, 30≤DI < 50 is susceptible to clubroot, and ≥50 is susceptible to clubroot.

3. The method for breeding clubroot-resistant Brassica napus according to claim 1, characterized in that: In S1, the selection of clubroot-resistant rapeseed lines specifically comprises the following steps: Step 1: Select pure line materials with different genetic backgrounds and specific traits from the Brassica napus germplasm resource bank as the first germplasm; Step 2: Identify the first germplasm for resistance to clubroot disease, and use the germplasm with a clubroot disease index of less than 30 as the second germplasm; Step 3: The second germplasm is identified for resistance to clubroot, and the second germplasm with a clubroot disease index of less than 15, excellent comprehensive agronomic traits and up-to-standard seed quality in six consecutive rounds of identification is selected as the third germplasm; Step 4: Using the third germplasm as a parent, artificially hybridizing different germplasms to obtain the first F1 generation of different hybrid combinations; Step 5: The first F1 generation is identified for its resistance to clubroot disease, and the parent combination corresponding to the first F1 generation with a clubroot disease index <5 is used as material for cultivating double haploid plants.

4. The method for breeding clubroot-resistant Brassica napus according to claim 3, characterized in that: In step 3, the comprehensive excellent agronomic traits refer to compact plant type, moderate plant height, early maturity and good fruit set.

5. The method for breeding clubroot-resistant Brassica napus according to claim 3, characterized in that: In step 3, the seed quality reaching the standard means that the seed oil content is greater than 40%, the seed glucosinolate content is less than 30 μmol / g cake, and the seed erucic acid content is less than 3%.

6. The method for breeding clubroot-resistant Brassica napus according to claim 4, characterized in that: In S1, inducing a double haploid plant population specifically includes: At the bolting and budding stage, the flower buds at the mononuclear marginal stage of the material for cultivating double haploid plants are taken for microspore culture. During the process of embryo formation, colchicine is added to double the chromosomes. After the embryos appear, they are differentiated and induced into seedlings to obtain a double haploid plant population.

7. The method for breeding clubroot-resistant Brassica napus according to claim 6, characterized in that: S5 also includes identification of resistance to Sclerotinia sclerotinia and identification standards for the incidence of Sclerotinia sclerotinia: Grade 0: no symptoms; Level 1: Less than 1 / 3 of the branches are diseased or the main stem has small lesions, and less than 1 / 4 of the siliques on the whole plant are affected; Level 2: 1 / 3-2 / 3 of the branches or the middle and upper part of the main stem are affected, and 1 / 4-2 / 4 of the siliques on the whole plant are affected; Level 3: more than 2 / 3 of the branches or the middle and lower part of the main stem are diseased, and 2 / 4-3 / 4 of the siliques on the whole plant are affected; Level 4: All or most of the branches are infected or there are lesions around the base of the main stem, and up to 3 / 4 of the siliques on the whole plant are infected. Select the candidate male and female hybrid combinations with the sclerotinia disease incidence level less than level 1.

8. The method for breeding clubroot-resistant Brassica napus according to claim 7, characterized in that: In S5, the sclerotinia disease incidence level of the clubroot-resistant Brassica napus variety is less than level 1.

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