Breeding method and application of brassica napus clubroot-resistant homogeneous recessive genic male sterility three-line

Abstract

The invention discloses a breeding method and application of a brassica napus clubroot-resistant homogeneous recessive genic male sterility three line, and belongs to the technical field of rape variety breeding. According to the method, a clubroot-resistant gene CRb and fertility-related genes * Ms3 / ms3 * and Rfb / Rfc are synchronously tracked through a molecular marker-assisted selection technology, a disease-resistant parent is taken as a donor to be hybridized with a recessive genic male sterile line, and a clubroot-resistant homogeneous dual-purpose line, a temporary maintainer line and a complete sterile line are rapidly bred by combining backcross and selfing strategies. The method solves the problems of non-homogeneity, low selection efficiency and long period of recessive three-line genic male sterility temporary maintainer lines and dual-purpose lines in traditional breeding of rape, significantly improves the breeding efficiency of anti-clubroot varieties and the uniformity of hybrids, and provides a technical guarantee for the safety of the rape industry.

Description

Technical Field

[0001] The present invention relates to the technical field of rapeseed variety breeding, in particular to a breeding method and application of a clubroot-resistant homogeneous recessive nuclear male sterile three-line of Brassica napus. Background Art

[0002] In recent years, rapeseed clubroot has become a major threat to the safety of the rapeseed industry. Clubroot is caused by the protist, root-knot fungus, which infects the roots. This fungus forms nodules on the roots, hindering nutrient and water absorption and, in severe cases, causing the entire plant to wither. Clubroot is a soil-borne disease, and its dormant spores can survive in the soil for over a decade. Furthermore, the fact that living root-knot fungi cannot be cultured in vitro makes its prevention and control a challenging area of ​​disease resistance research. Breeding disease-resistant varieties is the most cost-effective way to combat clubroot. Traditional breeding methods for developing clubroot-resistant varieties require inoculating intermediate materials with root-knot fungus each generation to determine whether they carry the gene for resistance. However, because root-knot fungi cannot be cultured in vitro, the type of fungus used for inoculation varies from generation to generation. Furthermore, the effectiveness of inoculation is significantly affected by the environment, significantly impacting the reliability of selection, consuming time and effort, and slowing down the breeding process.

[0003] The recessive three-line nuclear male sterility system of Brassica napus is an important way to utilize the heterosis of rapeseed. Many domestic breeding units use this system, such as Wanyou 14, Huayouza 50, and Zheyouza 1, which are all cultivated using this system. This system has stable sterility, flexible pairing, simple seed production technology, safe seed production, high yield, and low cost. The fertility of the recessive three-line nuclear male sterility system is controlled by a pair of sterility genes (Ms3 / ms3) and a pair of multiple alleles (Rf a / Rf b / Rf c ) interaction control, Ms3 is a wild-type fertile gene, and its recessive allele type ms3 is a sterile mutant. There are three alleles at the Rf locus, namely Rf a (Ms4), Rf b (Rf) and Rf c (rf), where Rf c is the wild-type fertility gene, Rf a is a mutant restorative fertility gene, and Rf b It is a mutant sterility gene, and the dominant and recessive relationship among the three is Rf a >Rf b >Rf c In the case of ms3 recessive homozygote (ms3ms3) and Rf locus is Rf b Rf b or Rf b Rf cUnder the same conditions, the plants showed male sterility; under other conditions, the plants showed normal male fertility. c Rf c The vast majority, with a very small number of genotypes being Ms3Ms3Rf b Rf b and Ms3Ms3Rf a Rf a , both are Rf c mutant genotype.

[0004] The recessive nuclear male sterile three-line is composed of dual-purpose lines (ms3ms3Rf b Rf b Sterile+Ms3ms3Rf b Rf b Fertile) and Linbao line (fertile ms3ms3Rf c Rf c ) composed of dual-purpose sterile plants and the protection line hybrid to produce a full sterile line (ms3ms3Rf b Rf c ), the fully sterile line and the restoration line are hybridized to produce commercial hybrid F1. Because of the generation of fully sterile populations, three-line matching is achieved in rapeseed hybrid seed production. At present, in the production application of this system, due to its complex multiple allelic genetic relationship, many front-line breeders still use traditional hybridization and test cross methods to select and breed dual-purpose lines and temporary protection lines respectively. The genetic backgrounds of the selected dual-purpose lines and temporary protection lines are different, and the hybrids produced are actually three-cross varieties. In this way, not only the uniformity of the hybrids is poor, but also the hybrid advantage will be reduced. Therefore, it is necessary to select and breed homogeneous dual-purpose lines and temporary protection lines. There are also some methods that use heat shock method to make fully sterile lines (ms3ms3Rf b Rf c ) to restore fertility and then isolate homogeneous CMS lines from the self-pollinated progeny. However, these methods still have limitations. First, they can only obtain CMS lines with similar backgrounds to existing CMS lines. Second, due to the heterozygous CMS lines (ms3ms3Rf b Rf c ) is a hybrid, and the LNP lines obtained through self-pollination are also in the process of segregation. Multiple generations of self-pollination of the heterozygous male sterile lines are also required, which is extremely time-consuming and labor-intensive, hindering the breeding process. Therefore, it is of great value to find solutions to the current problems in rapeseed production, such as the severe damage caused by clubroot, the difficulty in breeding recessive three-line nuclear male sterile lines, the heterogeneity of LNP lines and dual-purpose lines, the low uniformity of hybrids, and the reduction of heterosis. Summary of the Invention

[0005] The present invention aims to provide a simple breeding method and application for a clubroot-resistant homogeneous recessive genic male sterile tri-line in Brassica napus to address the aforementioned problems of the prior art. The present invention utilizes molecular marker-assisted selection (MASS) technology to simultaneously track clubroot-resistance genes and recessive genic male sterile fertility-related genes, enabling rapid generation of a genetically homogeneous clubroot-resistant tri-line after four to five generations of backcrossing.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a breeding method for a three-line homogeneous recessive male sterile line of Brassica napus resistant to clubroot, comprising breeding a disease-resistant parent C409R carrying the clubroot resistance gene CRb with a recessive male sterile dual-purpose line sterile plant ms3ms3Rf b Rf b Hybridization was performed to obtain the F1 generation; molecular markers were used to simultaneously screen the clubroot resistance gene CRb and the fertility-related genes Ms3 / ms3 and Rf b / Rf c , through backcrossing and selfing, and selecting RrMs3ms3Rf in each generation b Rf c The homogeneous disease-resistant line RRms3ms3Rf was bred by genotypic strategy. c Rf c and the homozygous disease-resistant dual-purpose line RRMs3ms3Rf b Rf b and RRms3ms3Rf b Rf b steps.

[0008] Furthermore, the molecular markers include:

[0009] The co-dominant molecular marker of the clubroot resistance gene CRb is A3-7, and its primer sequences are shown in SEQ ID NO.1 and NO.2;

[0010] The co-dominant molecular marker of the fertility-related gene *Ms3 / ms3* is Pms, and its primer sequences are shown in SEQ ID NO.3 and NO.4;

[0011] The fertility-related gene Rf b / Rf c The co-dominant molecular marker is A7-rf, and its primer sequences are shown in SEQ ID NO.5 and NO.6.

[0012] Furthermore, the Rf locus genotype of the donor parent was determined.

[0013] Furthermore, each backcross generation selected RrMs3ms3Rf b Rf cThe genotype individual plants were backcrossed, and the backcrossing was carried out for 4-5 generations.

[0014] Furthermore, the backcross population is no less than 80 plants.

[0015] Further select RrMs3ms3Rf b Rf c The genotype individual plants are self-pollinated, and the self-pollinated progeny population is no less than 300 plants.

[0016] Furthermore, the disease-resistant parent is C409R.

[0017] The present invention also provides a method for preparing a clubroot-resistant complete sterile line of Brassica napus, wherein the homogeneous disease-resistant line RRms3ms3Rf c Rf c The homozygous disease-resistant dual-purpose line RRMs3ms3Rf b Rf b and RRms3ms3Rf b Rf b The sterile plants of Brassica napus were hybridized to obtain a fully sterile line of Brassica napus resistant to clubroot.

[0018] The present invention also provides the use of the Brassica napus clubroot-resistant complete sterile line obtained by the preparation method in the breeding of rapeseed clubroot-resistant varieties.

[0019] The present invention discloses the following technical effects:

[0020] The present invention uses molecular marker-assisted selection technology to achieve synchronous tracking of clubroot-resistant genes and recessive nuclear sterile fertility-related genes, and can quickly obtain disease-resistant three-line lines with homogeneous genetic backgrounds after 4-5 generations of backcrossing. Indoor inoculation verification of disease-resistant strains shows that the disease index of the homozygous disease-resistant strain is only 8.7, which is highly resistant and significantly better than the susceptible control (78), which is highly susceptible. The present invention provides a simple, rapid and efficient breeding method for breeding excellent homogeneous three-line lines of cabbage rapeseed resistant to clubroot. By using this method, any excellent parent can be improved and transformed into a homogeneous nuclear sterile three-line line resistant to clubroot, solving the problem that rapeseed production is harmed by clubroot and that the recessive nuclear sterile three-line hybrids of cabbage rapeseed are three-cross varieties with poor uniformity and reduced heterosis, providing a material basis and technical guarantee for ensuring the production safety of the rapeseed industry. The method can be widely used in the breeding of disease-resistant varieties of cabbage rapeseed, and has both high efficiency, accuracy and practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 This is the electrophoresis diagram of PCR amplification of the clubroot resistance co-dominant marker A3-7 in a backcross population; the bands with large molecular weight are disease-resistant specific bands, 1-46 are the tested individual plants, 47 and 48 are disease-resistant (RR) and susceptible (rr) controls, respectively;

[0023] Figure 2 This is the electrophoresis diagram of PCR amplification of the recessive nuclear sterile allele locus Ms3 / ms3 in the segregating population; the band with a large molecular weight is the sterile genotype ms3ms3, 1-46 are the individual plants to be tested, and 47 and 48 are the Ms3Ms3 and Ms3ms3 genotype controls, respectively;

[0024] Figure 3 Recessive nuclear sterile allele locus Rf b / Rf c PCR amplification electrophoresis diagram of the isolated population; among them, 1-48 are the single strains to be detected, 49 and 50 are Rf b Rf c Genotype and Rf c Rf c genotype control;

[0025] Figure 4 The successful transformation of homogeneous clubroot-resistant emergency protection lines and dual-purpose lines;

[0026] Figure 5 For the technology roadmap;

[0027] Figure 6 These are the results of indoor inoculation of disease-resistant strains;

[0028] Figure 7 For each backcross generation, the proportion of disease-resistant and disease-susceptible plants was 50%.

[0029] Figure 8 The comparison between the successfully transformed sterile line and the original parent;

[0030] Figure 9 The rare genotype parent is Ms3Ms3Rf a Rf a Flow chart of the transfer technology. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1 Improvement of Clubroot Resistance in Recessive Genic Male Sterile Line 9012A of Brassica napus

[0037] 9012A is a recessive genic male sterile line of Brassica napus. It is stable and has good combining ability, but is highly susceptible to clubroot. c Rf c ), 9012A homozygous dual-purpose sterile line (ms3ms3Rf b Rf b ) as the female parent, hybrid, F1 generation (RrMs3Ms3Rf b Rf c ) Each gene locus is heterozygous, no screening is done, and F1 plants and 9012A Linbao line (rrms3ms3Rf c Rfc ) backcross; molecular markers were used to screen for fertility-related genes Ms3 / ms3 (Dun et al, 2011), Rf b / Rf c (GenBank: KX756984) and the clubroot resistance gene CRb (Hatakeyama et al., 2017). Using primer sequences and PCR reaction conditions, genotypes were calculated based on electrophoresis results. Molecular marker detection procedures were as follows: DNA from young leaves of parents and individual plants of each generation was extracted using the CTAB rapid extraction method. The clubroot resistance gene (RR) marker and fertility-related genes (Ms3 / ms3, Rf b / Rf c ) was labeled for PCR amplification. The PCR 10 μL universal reaction system contained 100 ng DNA template, 250 nmol L each of forward and reverse primers. -1 dNTP 0.25 nmol L -1 , Taq enzyme 1UμL -1 .

[0038] The clubroot resistance marker was screened using the co-dominant marker A3-7 (primer sequence as shown in SEQ ID NO. 1-2) within the CRb gene region (PCR reaction conditions were: 94°C pre-denaturation for 3 min; 94°C for 30 s, 55°C for 45 s, 72°C for 30 s, 35 cycles; 72°C for 5 min; storage at 4°C). The PCR product was electrophoresed on a 6% denaturing polyacrylamide (PAGE) gel at 70 W constant power for 50 min, followed by silver staining, development, and band analysis.

[0039] The nuclear sterility-related marker Ms3 / ms3 was screened using the co-dominant marker Pms (primer sequences are shown in SEQ ID NO.3-4) (PCR reaction conditions were: 94°C pre-denaturation for 3 min; 94°C for 30 s, 65°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 5 min; and storage at 4°C); the PCR products were electrophoresed on a 6% denaturing polyacrylamide (PAGE) gel at 70 W constant power for 1 h, followed by silver staining, development, and band analysis.

[0040] epistatic gene Rf b / Rf c The marker was screened with the co-dominant marker A7-rf (primer sequences are shown in SEQ ID NOs. 5-6) (PCR reaction conditions were: pre-denaturation at 94°C for 3 min; 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s; 72°C for 5 min; and storage at 4°C); the PCR products were electrophoresed on a 6% denaturing polyacrylamide (PAGE) gel at 70 W constant power for 45 min, followed by silver staining, development, and band analysis.

[0041] Primer pair A3-7 amplified two fragments of about 160 bp in size, of which the large molecular weight fragment was marked as 1, indicating the RR disease-resistant genotype, the small molecular weight fragment was marked as 2, indicating the rr disease-susceptible genotype, and the fragment with both large and small molecular weight was marked as 3, indicating the heterozygous Rr disease-resistant genotype ( Figure 1 The primer pair Pms amplified two fragments of about 200 bp in size. The large molecular weight fragment was marked as 1, which was the ms3ms3 genotype. The small molecular weight fragment was marked as 2, which was the Ms3Ms3 genotype. The fragments with both large and small molecular weights were marked as 3, which was the Ms3ms3 heterozygous genotype. Figure 2 ); A7-rf amplified two fragments with a molecular weight of 120 bp, of which the large molecular weight fragment was marked as 1, which was Rf c Rf c Genotype, small molecular weight fragment is recorded as 2, Rf b Rf b Genotype, both large and small molecular weight fragments are recorded as 3, which is a heterozygous genotype Rf b Rf c ( Figure 3 );

[0042] The primer sequences are as follows:

[0043] A3-7-F: 5'-CCTTACAAAGCTCATATACTT-3' (SEQ ID NO. 1);

[0044] A3-7-R: 5'-AGCCATCGTCCACAACTCG-3' (SEQ ID NO. 2);

[0045] Pms-F: 5'-GGATTTAGTGATGCAACACATG-3' (SEQ ID NO.3);

[0046] Pms-R: 5'-TGCGTATTCATCTGGTTCATCA-3' (SEQ ID NO. 4);

[0047] A7rf-F: 5'-CGGCCATTAGATAGGGCATT-3' (SEQ ID NO. 5);

[0048] A7rf-R: 5'-ACGATCCCAATCAGCTCAAC-3' (SEQ ID NO. 6).

[0049] Table 1 shows the genotype detection results of strain MR102 in the BC1 generation. There are 8 genotypes in the backcross generation. The probability of each genotype appearing is theoretically 1 / 8.b Rf c ) genotype single plant and the protection line (rrms3ms3Rf c Rf c ) backcross, and then each backcross generation was subjected to the same selection, and (RrMs3ms3Rf b Rf c ) plants and simultaneously screen for quality. To avoid plant damage from field pests and diseases and improper farming practices, each generation must ensure that 10 selected plants are present. Therefore, the population size for each backcross generation needs to be at least 80 plants.

[0050] Table 1 Genotype distribution of BC1 generation backcross individual plant offspring

[0051]

[0052]

[0053] Continuing and temporary insurance system (rrms3ms3Rf c Rf c ) After 4-5 generations of backcrossing, the background recovery rate has basically reached more than 95%, and about 10 RrMs3ms3Rf b Rf c The genotyped individual plants were self-pollinated. The self-pollinated seeds were sown in plug trays, and DNA was extracted from individual plants at the seedling stage to test the genotype. The goal was to screen out the homozygous disease-resistant line (RRms3ms3Rf c Rf c ) and homozygous disease-resistant dual-purpose lines (RRMs3ms3Rf b Rf b +RRms3ms3Rf b Rf b ) These three genotypes. b Rf c Self-pollination of a single genotype plant will produce 27 genotypes. The probability of the three target genotypes we need is 1 / 64. Therefore, in order to ensure that all target genotypes can be selected and seeds can be harvested, it is best to have more than 5 plants in each selected plant, which requires RrMs3ms3Rf b Rf c The self-pollinated offspring of the genotype have a population of more than 300 individual plants.

[0054] The line MR102 was self-pollinated to produce 204 plants, from which 7 target lines with homozygous disease resistance were selected: 2 homozygous resistant to Linbao, 3 homozygous resistant to dual-purpose fertile lines (RRMs3ms3Rf b Rf b ), two homozygous resistant dual-purpose sterile plants (RRms3ms3Rf b Rfb ).

[0055] To verify the effect of molecular markers on the selection of disease-resistant genes, two lines were selected for indoor inoculation. The genotypes of the two lines were heterozygous disease-resistant Rr and homozygous disease-resistant RR, respectively. The control was the Linbao line (rr) without disease-resistant genes. The results of indoor inoculation are shown in Tables 2 and Figure 6 .

[0056] Table 2 Statistics of indoor root knot fungus inoculation results

[0057]

[0058] Among them, the grading standards for levels 0-3 are:

[0059] Grade 0: No symptoms of infection, normal root system; Grade 1: No nodules on the taproot, with a few diffuse, small nodules on the lateral roots; Grade 2: Medium-sized nodules on the taproot and lateral roots; Grade 3: The diameter or length of the taproot tumor is at least three times the diameter of the stem base, or the tumor is ulcerated. Disease rate (%) = number of diseased plants / total number of plants surveyed × 100; disease index = ∑(number of diseased plants × corresponding grade) / (total number of plants surveyed × highest grade) × 100. A disease index of 0 indicates immunity (I); an index greater than 0 and less than or equal to 10 indicates high resistance (HR); an index greater than 10 and less than or equal to 30 indicates resistance (R); an index greater than 30 and less than or equal to 50 indicates susceptible (S); and an index greater than 50 indicates highly susceptible (HS).

[0060] As can be seen from Table 2, the disease rate of the susceptible control reached 97.9%, with a disease index of 78, indicating high susceptibility; the disease rate of the homozygous resistant strain MR63 was greatly reduced, with only one plant showing grade 3 susceptibility, and a disease index of 8.7, indicating high resistance; the heterozygous resistant strain MR45 had a higher incidence rate than the homozygous resistant strain and lower than the susceptible control, as it separated 1 / 4 of the susceptible genotypes, which was in line with expectations ( Figure 6 ).

[0061] Two homozygous disease-resistant Linbao lines were bagged for self-pollination; at the same time, three homozygous resistant dual-purpose fertile plants and two sterile plants were transplanted together, pollinated with tents, and seeds from the sterile plants were harvested to achieve dual-purpose line reproduction (the sterile line 9012A was improved into the homogeneous resistant sterile line 9012AR carrying the clubroot resistance gene CRb (for technical details, see Figure 5 )). Subsequent expansion and reproduction, Figure 4 In order to expand the reproduction of homogeneous disease-resistant temporary protection lines and dual-purpose lines, it can be seen that the temporary protection lines and dual-purpose lines are almost identical in agronomic traits. By using the disease-resistant homozygous dual-purpose line sterile plants and the disease-resistant temporary protection lines, disease-resistant fully sterile lines can be prepared, and the fully sterile lines are used for commercial hybrid seed grouping.

[0062] Example 2: Transforming the superior parent ZA39 into a recessive genic male sterile line resistant to clubroot

[0063] ZA39 is an excellent parent with high combining ability, resistance to sclerotinia rot, resistance to lodging, and high oil content, but it is highly susceptible to clubroot. In order to better utilize the parent combination, the method of the present invention is used to transform it into a recessive nuclear male sterile three-line resistant to clubroot.

[0064] The ZA39Rf locus genotype was detected using marker A7-rf, which was wild-type Rf c Rf c Genotype. With C409R containing the clubroot resistance gene CRb as the male parent, 9012A homozygous dual-purpose sterile line (ms3ms3Rf b Rf b ) as the female parent (any homozygous sterile plant of the recessive three-line nuclear sterile dual-purpose line can be selected here), hybridize, and all gene loci of the F1 generation are heterozygous (RrMs3ms3Rf b Rf c ), without screening. Select single plants and superior parent ZA39 (rrMs3Ms3Rf c Rf c ) backcross, and molecular markers were used to screen fertility-related genes (Ms3 / ms3 and Rf b / Rf c ) and clubroot resistance gene (CRb). The BC1 generation will also have 8 different genotypes, half of which carry the clubroot resistance gene and show half resistance to clubroot ( Figure 7 ), different from Example 1 of the present invention, these 8 genotypes are all fertile phenotypes. The genotype distribution of BC1 generation backcross single plant offspring is shown in Table 3. Our target genotype (RrMs3ms3Rf b Rf c ) also has a 1 / 8 probability of occurrence. Similarly, to ensure that the target individual plant is selected and seeds are received, a population of about 80 backcross offspring is required.

[0065] The subsequent specific screening steps are the same as in Example 1, and each generation selects (RrMs3ms3Rf b Rf c ) genotype single plant continues to be the superior parent ZA39 (rrMs3Ms3Rf c Rf c ) backcross. Unlike the sterile line in Case 1, all intermediate materials are fertile genotypes. After 5 generations of backcross, (RrMs3ms3Rf b Rf c ) genotype single plants were self-pollinated. Among them, the line ZM103 was self-pollinated to produce 256 single plants, and the homozygous disease-resistant line (RRms3ms3Rf c Rf c) 4 strains, homozygous disease-resistant dual-purpose fertile plants (RRMs3ms3Rf b Rf b )5 strains, sterile strain (RRms3ms3Rf b Rf b ) 6 plants, the Linbao line is self-fertilized, and the dual-purpose line is sister-fertilized. The sterile line that was bred was completely sterile ( Figure 8 ), with petals slightly smaller than those of the original parent, ZA39, while all other agronomic traits were consistent with the original parent. Indoor clubroot inoculation also revealed that the successfully transformed sterile line had a disease rate of 16% and a disease index of 7.9, indicating high resistance, while the original parent had a disease rate of 97% and a disease index of 90, indicating high susceptibility. Subsequent propagation was expanded, and the newly developed clubroot-resistant sterile line was named 9022RA.

[0066] Table 3 Genotype distribution of BC1 generation backcross individual plant offspring

[0067]

[0068] It is worth noting that if the parent of the transfer happens to be Ms3Ms3Rf, which has a very low probability of occurrence a Rf a Genotype, a complex breeding procedure is required. The specific steps are shown in Figure 9 , it can also be transformed into a homogeneous nuclear male sterile three-line resistant to clubroot disease.

[0069] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for breeding a clubroot-resistant homogeneous recessive male sterile three-line of Brassica napus, characterized in that: The method comprises the following steps: combining a disease-resistant parent carrying the clubroot resistance gene CRb with a recessive nuclear male sterile dual-purpose line sterile plant ms3ms3Rf b Rf b Hybridization to obtain F1 generation; Simultaneous screening of clubroot resistance gene CRb and fertility-related genes *Ms3 / ms3* and Rf using molecular markers b / Rf c By backcrossing and self-pollination, the homogeneous disease-resistant protection lines RRms3ms3Rf c Rf c and the homozygous disease-resistant dual-purpose line RRMs3ms3Rf b Rf b and RRms3ms3Rf b Rf b steps.

2. The breeding method according to claim 1, characterized in that: The molecular markers include: The co-dominant molecular marker of the clubroot resistance gene CRb is A3-7, and its primer sequences are shown in SEQ ID NO.1 and NO.2; The co-dominant molecular marker of the fertility-related gene *Ms3 / ms3* is Pms, and its primer sequences are shown in SEQ ID NO.3 and NO.4; The fertility-related gene Rf b / Rf c The co-dominant molecular marker is A7-rf, and its primer sequences are shown in SEQ ID NO.5 and NO.

6.

3. The breeding method according to claim 1, characterized in that: Each backcross generation selected RrMs3ms3Rf b Rf c The genotype individual plants were backcrossed, and the backcrossing was carried out for 4-5 generations.

4. The breeding method according to claim 1, characterized in that: The backcross population is no less than 80 plants.

5. The breeding method according to claim 1, characterized in that: Select RrMs3ms3Rf b Rf c The genotype individual plants are self-pollinated, and the self-pollinated progeny population is no less than 300 plants.

6. The breeding method according to claim 1, characterized in that: The disease-resistant parent is C409R.

7. A method for preparing a clubroot-resistant male sterile line of Brassica napus, characterized in that: The homogeneous disease-resistant protective system RRms3ms3Rf described in claim 1 c Rf c The homozygous disease-resistant dual-purpose line RRMs3ms3Rf b Rf b and RRms3ms3Rf b Rf b The sterile plants of Brassica napus were hybridized to obtain a fully sterile line of Brassica napus resistant to clubroot.

8. Use of the clubroot-resistant complete sterile line of Brassica napus obtained by the preparation method according to claim 7 in the breeding of clubroot-resistant rapeseed varieties.

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