Molecular marker combination for identifying high-clubroot-resistance material of Chinese cabbage and application of molecular marker combination

Abstract

The invention provides a molecular marker combination for identifying a Chinese cabbage high-clubroot-resistance material and application of the molecular marker combination, and belongs to the technical field of Chinese cabbage breeding. The molecular marker combination is composed of a CRb molecular marker B0902 and a Crr3 molecular marker OPC11-2S of a Chinese cabbage No.3 linkage group and a molecular marker P29 of a Chinese cabbage No.8 linkage group. The molecular marker combination not only can screen high-clubroot-resistance Chinese cabbage varieties on the market, but also can rapidly breed high-clubroot-resistance breeding materials aggregating three disease-resistant sites, and can be effectively applied to screening or breeding of high-clubroot-resistance varieties or materials.

Description

Technical Field

[0001] The invention belongs to the technical field of Chinese cabbage breeding, and in particular relates to a molecular marker combination for identifying Chinese cabbage high-resistance to clubroot disease materials and an application thereof. Background Art

[0002] Crucifer clubroot is a worldwide disease caused by Plasmodiophora brassica Woronin. It has a wide host range and can harm more than 100 cultivated and wild cruciferous plants, including cabbage, kale, radish, cauliflower, rapeseed, etc. Chinese cabbage is one of the cruciferous vegetables most seriously affected by clubroot. In recent years, clubroot has occurred in most parts of my country, including coastal areas, Yunnan, Guizhou, Sichuan, parts of the northeast, and western regions. The area of ​​occurrence has increased dramatically and the severity has increased year by year. It has become the main disease in the main cabbage producing areas.

[0003] Since the dormant spores of the root-root pathogen have strong stress resistance and can survive in the soil for more than 10 years, chemical control is extremely difficult. The selection and application of disease-resistant varieties is the fundamental way to solve the harm of root-root disease. Molecular marker-assisted breeding is an effective means to breed Chinese cabbage varieties resistant to root-root disease. At present, there are many gene loci for cabbage resistance to root-root disease reported in research, such as Crr2 on the A1 linkage group, CRc and Rcr8 on the A2 linkage group, Crr3, CRa, CRb, CRk, CRd, Rcr1, Rcr2, Rcr4 on the A3 linkage group, CrrA5 on the A5 linkage group, Crr4 on the A6 linkage group, Crr1 (Crr1a, Crr1b), Rcr9, CRs on the A8 linkage group, etc. However, most of the molecular markers of the reported gene loci cannot well identify the disease resistance of varieties and materials (the marker results are inconsistent with the inoculation identification results), and it is impossible to efficiently screen out varieties or materials with high resistance to root-root disease. Summary of the invention

[0004] The present invention provides a molecular marker combination for identifying Chinese cabbage highly resistant to clubroot and an application thereof. The molecular marker combination can not only screen out Chinese cabbage varieties with high resistance to clubroot on the market, but also quickly breed highly resistant to clubroot breeding materials that aggregate at least two or more disease-resistant sites, and can be effectively applied to the screening or breeding of highly resistant to clubroot varieties or materials.

[0005] In order to achieve the above object, the present invention provides a molecular marker combination for screening Chinese cabbage materials with high resistance to clubroot, characterized in that it consists of CRb molecular marker B0902 of linkage group 3 of Chinese cabbage, Crr3 molecular marker OPC11-2S and molecular marker P29 of linkage group 8.

[0006] Preferably, the nucleotide sequence of the forward primer for amplifying the molecular marker B0902 is shown in SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2;

[0007] The nucleotide sequence of the forward primer for amplifying the molecular marker OPC11-2S is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 4;

[0008] The nucleotide sequence of the forward primer for amplifying the molecular marker P29 is shown in SEQ ID NO:5, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:6.

[0009] The present invention also provides a kit for screening Chinese cabbage materials with high resistance to clubroot, comprising the primer pair described in the above technical solution.

[0010] The present invention provides an application of the molecular marker combination according to the above technical solution, the primer pair according to the above technical solution or the kit according to the above technical solution in screening Chinese cabbage varieties or materials with high resistance to clubroot disease, or breeding high clubroot disease-resistant breeding materials with three disease resistance sites.

[0011] Preferably, the screening comprises the following steps:

[0012] Using the primers described in claim 2 to perform PCR amplification on the Chinese cabbage variety or material sample to be screened to obtain a PCR product;

[0013] The obtained PCR products are used for agarose gel electrophoresis or SDS-PAGE gel electrophoresis, and samples of varieties or materials having disease-resistant characteristic bands of three molecular markers are judged as varieties or materials of Chinese cabbage with high resistance to clubroot.

[0014] Preferably, the disease resistance characteristic band of molecular marker B0902 is 160 bp, the disease resistance characteristic band of molecular marker OPC11-2S is 1300 bp, and the disease resistance characteristic band of molecular marker P29 is 77 bp.

[0015] Preferably, the disease resistance of the variety or material having two disease resistance characteristic bands is generally better than that of the variety or material having only one disease resistance band;

[0016] When the disease resistance bands of OPC11-2S and P29 are homozygous disease resistance bands, the disease resistance is better than that when B / O or B / P are both heterozygous.

[0017] Preferably, the reaction procedure of PCR amplification is:

[0018] The PCR program for B0902 was pre-denaturation at 95°C for 4 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 20 s, 30 cycles, and extension at 72°C for 7 min;

[0019] The PCR program for OPC11-2S was pre-denaturation at 95.5°C for 4 min, denaturation at 95°C for 30 s, annealing at 57°C for 60 s, extension at 72°C for 60 s, 35 cycles, and extension at 72°C for 10 min;

[0020] The reaction program for PCR amplification of P29 was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 s, annealing at 54°C for 30 s, extension at 72°C for 45 s, 35 cycles, and extension at 72°C for 10 min.

[0021] Preferably, the reaction system for PCR amplification is:

[0022] The reaction volume of the PCR mixture was 10 μL, 2×Tag Plus Master Mix 5 μL, 10 mmol·μL -1 0.5 μL and 10 ng μL of primers -1 4 μL of DNA template.

[0023] The present invention provides a method for breeding Chinese cabbage varieties or materials with high resistance to clubroot, comprising the following steps:

[0024] The cabbage varieties or materials are screened for clubroot resistance using the molecular marker combination for screening cabbage high clubroot resistance materials described in the above technical solution, and breeding materials having disease resistance characteristic bands of three molecular markers in the molecular marker combination are selected, namely, the high clubroot resistance breeding material CCR108-47.

[0025] Compared with the prior art, the advantages and positive effects of the present invention are:

[0026] The present invention uses a molecular marker combination of CRb molecular marker B0902 of Chinese cabbage linkage group 3, Crr3 molecular marker OPC11-2S and molecular marker P29 of linkage group 8 to identify clubroot resistance in Chinese cabbage varieties or materials. The combined application of the three molecular markers can not only screen out Chinese cabbage varieties with high clubroot resistance on the market, but also quickly breed high clubroot resistance breeding materials that aggregate at least two or more disease resistance sites, and can be effectively applied to the screening or breeding of high clubroot resistance varieties or materials.

[0027] The present invention further screened out a highly clubroot-resistant breeding material CCR108-47 using the above-mentioned molecular marker combination, which contains three homozygous disease-resistant major effect genes. On the one hand, it can be used to directly formulate a highly clubroot-resistant combination, and on the other hand, it can be used as a clubroot-resistant breeding source to breed other excellent clubroot-resistant breeding materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The electrophoresis band results of the marker identification of 30 materials B0902. M: standard band, 1-30 are test varieties, the lower band is the disease resistance characteristic band 160bp; 1, 2, 3, 4, 6, 7, 8, 13-22, 25, 27, 28, a total of 20 materials have disease resistance characteristic bands;

[0029] Figure 2 The electrophoresis band results of 30 materials OPC11-2S marker identification. M: standard band, 1-30 are test varieties, the upper band is the disease resistance characteristic band 1300bp; 6 materials 6, 7, 8, 15, 22, 25 have disease resistance characteristic bands;

[0030] Figure 3 The electrophoresis band results of P29 marker identification of 30 materials. The upper band is a disease resistance characteristic band of 77bp; 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 17, 24, 26, 29, a total of 14 materials have disease resistance characteristic bands;

[0031] Figure 4 The three disease resistance loci were all homozygous for the disease resistance material CCR108-47 by using the combination of three molecular markers;

[0032] Figure 5 It is a breeding material with high resistance to clubroot disease, CCR108-47;

[0033] Figure 6 The results of inoculation of CCR108-47 against 8 strains of clubroot pathogens are shown in Figure 2. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1

[0036] The research team used 11 molecular markers to test 128 clubroot-resistant heading and non-heading cabbage varieties from home and abroad. Among them, markers SC2930-T-FW / SC2930-RV, SC2930-Q-FW / SC2930-RV, CRaim1 / CRami2 are linked to the disease resistance gene CRa (Matsumoto et al., 2012; Aruga et al., 2013), marker B0902 is linked to the gene CRbkato (Kato et al., 2013), marker TCR05 is linked to CRbPiao (Piao et al., 2004), marker B50-C9-FW / B50-6R-FW is linked to the gene CRc (Matsumoto et al., 2012), marker HC688-6-RV / HC688-7-RV is linked to the gene CRk (Matsumoto et al., 2012), marker BRMS-088 and BRMS-096 are linked to genes Crr1 and Crr2, respectively (Suwabe et al., 2013). al., 2003), marker OPC11-2S linked to Crr3 (Saito et al., 2006), and marker P29 newly developed by our research group and linked to the clubroot resistance trait of Degao CR117 (research group patent). All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The reaction volume of the PCR mixture was 10 μL, 2×Tag PlusMaster Mix 5 μL, 10 mmol·μL -1 0.5 μL and 10 ng μL of primers -1 4 μL of DNA template was added, and the insufficient part was supplemented with ddH2O water, and amplified using ABI Veriti gradient PCR instrument. The PCR product was electrophoresed on agarose or 6% non-denaturing polyacrylamide gel at constant power for 1-1.5 h, stained with simple silver staining method, developed, read with the help of a light box, and then photographed and archived.

[0037] Results: The most common varieties among the 128 varieties were CRk disease resistance bands, which were 112. Only 16 materials did not have CRk disease resistance bands, and 10 of them were CR117-like varieties. The second largest number of varieties were B0902 disease resistance bands, which were 96. The disease resistance marker CRaim1 of the CRa gene was consistent with the disease resistance band of B0902. The disease resistance marker Sc2930-T of the CRa gene was inconsistent with B0902 in 5 varieties, and the consistency between the two was 94.4%. This showed that CRa and CRbkato were closely linked or the same gene, and the B0902 marker was stable and co-dominant, with the best marker effect. Among the 96 varieties, 92 varieties also had CRk disease resistance bands. Among the 128 varieties, 32 varieties had CR117 (P29) disease resistance markers, and only 4 varieties had Crr3 disease resistance markers, and these 4 varieties also had B0902 disease resistance bands. There are 8 varieties with both B0902 and P29 resistance bands. Among the 128 varieties, 8 varieties are susceptible to the disease with both B0902 and P29 markers. Most of these varieties have the disease resistance gene CRk or CRC, and they are basically susceptible to the disease in production. There is no polymorphism between the markers TCR05, BRMS-088 and BRMS-096. The previous inoculation identification results of our research group showed that varieties with the Crr3 resistance marker (and the CRbkato resistance marker) and varieties with both the B0902 resistance band and the P29 resistance band have strong disease resistance.

[0038] Most of the disease resistance sites on Chinese cabbage are concentrated on chromosomes A03 and A08, with 10 and 8 sites respectively. In addition, a few disease resistance sites have been located on chromosomes A01, A02, and A06. Although each disease resistance site has a corresponding molecular marker, most molecular markers have poor universality due to the different research materials used by researchers, and there are very few markers that can be used to assist breeding screening. In many years of breeding work for resistance to clubroot, our research group screened related molecular markers retrieved from literature and found that only some markers closely linked to the disease resistance gene sites CRa (CRb) and Crr3 can effectively mark the clubroot resistance of different materials. Based on the screening of a large number of molecular markers, a new effective marker was developed (Indel marker P29 located on chromosome A08 and closely linked to the disease-resistant variety CR117) (Zhang Qingxia et al., 2019 Journal of Horticulture Sinica Supplement). The most representative effective markers screened or developed by the research team include: molecular marker B0902, which is tightly linked to the disease resistance locus CRa / CRb on chromosome A03; molecular marker OPC11-2S, which is tightly linked to the disease resistance gene locus Crr3 on chromosome A03; and marker P29, which is tightly linked to the clubroot resistance trait of Degao CR117 on chromosome A08. These three markers are widely used in the screening of disease-resistant materials.

[0039] The disease resistance markers CRa, B0902, TCR74, OPC11-2S, HC688-6-R on chromosome 3, Crr1a, P29, BRMS-088 on chromosome 8, and BRMS-096 on chromosome 2, a total of 9 molecular markers, were used to identify the disease resistance genes of the 30 materials inoculated and identified. The results are shown in Table 1.

[0040] Table 1 The results of labeling 30 materials using 9 molecular markers

[0041]

[0042]

[0043] It can be seen from the marker results in Table 1 that the marker results of CRa and B0902 are basically the same, and the two can replace each other. The four markers TCR74, BRMS-088, BRMS-096, and HC688-6-R have bands in both susceptible controls and resistant varieties, and cannot be used as markers for disease resistance identification. The marker results of Crr1a and P29 are mostly consistent, but P29 is more accurate. For example, materials No. 3, No. 7, and No. 5 are varieties bred by the research group. The disease resistance locus on chromosome 8 should be heterozygous, but it was identified as homozygous disease resistance using the Crr1a marker. Therefore, B0902 (which can be replaced by CRa), P29, and OPC11-2S are markers of three major disease resistance loci. The combination of the three markers can select highly resistant varieties or breeding materials with three major disease resistance loci. Therefore, the present invention provides a molecular marker combination for screening Chinese cabbage materials with high resistance to clubroot, which consists of the CRb molecular marker B0902 of linkage group 3 of Chinese cabbage, the Crr3 molecular marker Opc11-2s and the molecular marker P29 of linkage group 8.

[0044] Example 2

[0045] The screening of Chinese cabbage materials with high resistance to clubroot disease includes the following steps:

[0046] Firstly, the PCR amplification is performed on the Chinese cabbage varieties or material samples to be screened using primers for amplifying molecular markers to obtain PCR products;

[0047] The obtained PCR products are used for SDS-PAGE gel electrophoresis, and samples of varieties or materials having disease-resistant characteristic bands of three molecular markers are judged as varieties or materials of Chinese cabbage with high resistance to clubroot.

[0048] Specifically, the nucleotide sequence of the forward primer for amplifying the molecular marker B0902 is shown in SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2;

[0049] The nucleotide sequence of the forward primer for amplifying the molecular marker OPC11-2S is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 4;

[0050] The nucleotide sequence of the forward primer for amplifying the molecular marker P29 is shown in SEQ ID NO:5, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:6.

[0051] In a preferred embodiment, the reaction procedure of PCR amplification is:

[0052] The PCR program for B0902 was pre-denaturation at 95°C for 4 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 20 s, 30 cycles, and extension at 72°C for 7 min;

[0053] The PCR program for OPC11-2S was pre-denaturation at 95.5°C for 4 min, denaturation at 95°C for 30 s, annealing at 57°C for 60 s, extension at 72°C for 60 s, 35 cycles, and extension at 72°C for 10 min;

[0054] The reaction program for PCR amplification of P29 was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 s, annealing at 54°C for 30 s, extension at 72°C for 45 s, 35 cycles, and extension at 72°C for 10 min.

[0055] In a preferred embodiment, the reaction system for PCR amplification is:

[0056] The reaction volume of the PCR mixture was 10 μL, 2×Tag Plus Master Mix (Novozyme) 5 μL, 10 mmol·μL -1 0.5 μL of each primer (Shanghai Biotechnology Co., Ltd.) and 4 μL of 10 ng·μL-1 DNA template.

[0057] In a preferred embodiment, the PAGE gel electrophoresis conditions are:

[0058] The PCR product of primer P29 was electrophoresed by 6% non-denaturing polyacrylamide gel electrophoresis at a voltage of 2000 V, a current of 60 mA, a power of 50 W, and a constant power electrophoresis time of 1 h.

[0059] The PCR product of primers B0902 and OPC11-2S was electrophoresed with 100 ml of 1% agarose and 10 μL of 1000×Ultre GelRed (Novagen), voltage 170 V, current 300 mA, and electrophoresis time 20 min.

[0060] Example 3

[0061] Technical example: From May to June 2024, 30 Chinese cabbage varieties (materials) with different resistances were inoculated with different clubroot pathogens, and DNA was extracted for molecular marker identification. The test results are shown in Table 2.

[0062] Table 2 Results of three molecular markers and disease index of 30 materials for inoculation identification

[0063]

[0064]

[0065] Note: Molecular marker results: H: heterozygous disease resistance band, R: homozygous disease resistance band, there are many physiological races of clubroot disease, and the disease resistance gene has different resistance to different physiological races.

[0066] Resistance to 4 types of pathogens: HR (high resistance): disease index ≤11.1, R (resistant): disease index ≤33.3, M (medium resistance): disease index ≤55.5, S (susceptible): disease index >55.5.

[0067] like Figure 1-3 Combined with the results of the disease index of inoculation identification in Table 2, both materials No. 6 and No. 7 contain three disease resistance characteristic bands at the same time, and the inoculation identification results show high resistance or disease resistance to four pathogens from different sources. No. 2, 3, 4, and 17 contain two disease resistance markers (B and P), and their overall disease resistance is better than that of materials No. 1, 13, 14, 19, 16, 18, 20, 21, 27, 28 and No. 9, 10, 11, 12, 24, and 26, which only contain one disease resistance marker. No. 8, 15, 22, and 25 contain two disease resistance markers (B and O), and their overall disease resistance is also better than that of materials No. 1, 13, 14, 19, 16, 18, 20, 21, 27, and 28, which only contain one disease resistance marker. Material No. 29, P29, is a homozygous disease resistance band, and its disease resistance is significantly better than materials No. 9, 10, 11, 12, 24, 26, etc., where P29 is a heterozygous band, and is also better than materials No. 2, 3, 4, 17, which contain two disease resistance markers (B and P). When the OPC11-2S disease resistance band is homozygous, its disease resistance is also significantly better than the OPC heterozygous band, and is also better than the two heterozygous bands of B and O (not reflected in this test result, but has been proven by previous tests of the research group). When B0902 is a homozygous disease resistance band, the disease resistance is not improved in most cases, such as No. 1, 13, 14, 19, 20, and No. 16, 18, 21. The disease resistance of No. 27 and No. 28 (B0902 homozygous disease resistance band) is better than the above 8 materials, and it is possible that they contain other unknown disease resistance genes.

[0068] From the above, it can be seen that the molecular marker combination provided in the present application can be effectively used for the screening of Chinese cabbage materials with high resistance to clubroot. By using molecular markers and combining them with disease resistance gene identification, it can be seen that the disease resistance of varieties or materials with two disease resistance characteristic bands is generally better than that of varieties or materials with only one disease resistance band; when the Opc11-2s and P29 disease resistance bands are homozygous disease resistance bands, the disease resistance is better than that of heterozygous bands.

[0069] Example 4

[0070] Based on the above method, the Chinese cabbage high resistance to clubroot disease material CCR108-47 (such as Figure 5 The breeding method is as follows:

[0071] AQ-5-7 (breeding material selected by the research team and identified as BROR by molecular markers) was hybridized with BY17-5 (breeding material selected by the research team and identified as PR by molecular markers). The F1 generation (Qingyan CCR108BHOHPH) was inoculated and identified to be highly resistant to clubroot pathogens from various regions.

[0072] In March 2021, Qingyan CCR108 (F1 generation) seedlings were vernalized → In July, the immature seeds of F1 self-pollination (F2 generation) were stripped of embryos and germinated and vernalized at low temperature (July 26) → Sowing in an artificial climate chamber on August 23 → Molecular identification and screening of F2 plants in September → The 108-47 plant was genotype B for resistance to clubroot R O R P R (3R)→2022.6 obtained the self-pollinated seeds (F3 generation) of 108-47 (confirmed as 3R by molecular markers), inoculated and identified the clubroot in the autumn of 2022 (identification number 15) and observed the field planting (field number 1078). The inoculated and identified 108-47 F3 generation showed high immune resistance to 7 root-knot fungi from Qingdao, Chongqing, Yunnan, Guizhou, Hebei and other places (1 Qingdao Academy of Agricultural Sciences 2015, 2 Qingdao Licha Liangxiang 2019, 3 Chongqing Wulong 2020, 4 Yunnan Kunming 2019, 5 Yunnan Shuanglong 2013, 7 Guizhou Bijie 2020, 8 Hebei Tangshan 2021). It only showed moderate resistance to root-knot fungi No. 6 (Huoshaoping, Hubei 2020, the most pathogenic pathogen known so far). Figure 6 In 2023, self-pollination and field selection will continue, and the 108-47 will be sown in spring 2024 with stable traits. This CCR108-47 contains three homozygous disease resistance major effect genes (such as Figure 4 As shown in the figure), on the one hand, it can be used to directly formulate a combination with high resistance to clubroot disease, and on the other hand, it can be used as a source of clubroot disease resistance to breed other excellent clubroot disease resistance breeding materials.

Claims

1. A molecular marker combination for screening Chinese cabbage materials with high resistance to clubroot, characterized in that: It consists of the CRb molecular marker B0902 of linkage group 3, the Crr3 molecular marker OPC11-2S and the molecular marker P29 of linkage group 8 of Chinese cabbage.

2. A primer pair for amplifying the molecular marker combination according to claim 1, characterized in that: The nucleotide sequence of the forward primer for amplifying the molecular marker B0902 is shown in SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2; The nucleotide sequence of the forward primer for amplifying the molecular marker OPC11-2S is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 4; The nucleotide sequence of the forward primer for amplifying the molecular marker P29 is shown in SEQ ID NO:5, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:

6.

3. A kit for screening Chinese cabbage materials with high resistance to clubroot, characterized in that: Comprising the primer pair described in claim 2.

4. Use of the molecular marker combination according to claim 1, the primer pair according to claim 2 or the kit according to claim 3 in screening Chinese cabbage varieties or materials with high resistance to clubroot, or in breeding high resistance to clubroot breeding materials with three disease resistance sites.

5. The use according to claim 4, characterized in that: The following steps are involved in the screening process: Using the primers described in claim 2 to perform PCR amplification on the Chinese cabbage variety or material sample to be screened to obtain a PCR product; The obtained PCR products are used for agarose gel electrophoresis or SDS-PAGE gel electrophoresis, and samples of varieties or materials having disease-resistant characteristic bands of three molecular markers are judged as varieties or materials of Chinese cabbage with high resistance to clubroot.

6. The use according to claim 5, characterized in that: The disease resistance characteristic band of molecular marker B0902 is 160bp, the disease resistance characteristic band of molecular marker OPC11-2S is 1300bp, and the disease resistance characteristic band of molecular marker P29 is 77bp.

7. The use according to claim 6, characterized in that: The disease resistance of varieties or materials with two disease resistance characteristic bands is generally better than that of varieties or materials with only one disease resistance band; When the disease resistance bands of OPC11-2S and P29 are homozygous disease resistance bands, the disease resistance is better than that when B / O or B / P are both heterozygous.

8. The use according to claim 5, characterized in that: The reaction procedure for PCR amplification is: The PCR program for B0902 was pre-denaturation at 95°C for 4 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 20 s, 30 cycles, and extension at 72°C for 7 min; The PCR program for OPC11-2S was pre-denaturation at 95.5°C for 4 min, denaturation at 95°C for 30 s, annealing at 57°C for 60 s, extension at 72°C for 60 s, 35 cycles, and extension at 72°C for 10 min; The reaction program for PCR amplification of P29 was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 s, annealing at 54°C for 30 s, extension at 72°C for 45 s, 35 cycles, and extension at 72°C for 10 min.

9. The use according to claim 5, characterized in that: The reaction system for PCR amplification is: The reaction volume of the PCR mixture is 10 μL, 2×Tag Plus Master Mix 5 μL, 10 mmol μL -1 0.5 μL and 10 ng μL of primers -1 4 μL of DNA template.

10. A method for breeding Chinese cabbage varieties or materials with high resistance to clubroot, characterized in that: The following steps are involved: The clubroot resistance screening of Chinese cabbage varieties or materials is performed using the molecular marker combination for screening Chinese cabbage high clubroot resistance materials as described in claim 1, and breeding materials having disease resistance characteristic bands of three molecular markers in the molecular marker combination are selected, which are breeding materials with high clubroot resistance.

Images