Mapping of a pair of dominant complementary genes resistant to clubroot in Chinese cabbage and tightly linked SSR molecular markers and their applications

Abstract

The present invention discloses the mapping of a pair of dominant complementary clubroot-resistant genes in Chinese cabbage and closely linked SSR molecular markers and their applications, belonging to the fields of crop breeding and biomolecular genetics. In the present invention, the disease-resistant material 'CR-20' is crossed with the disease-susceptible material 'BJN3-1', and the disease-resistant genes contained in the disease-resistant material are preliminarily mapped by constructing an F2 generation genetic segregation population. By adopting the method of genetic map mapping combined with BSA sequencing, the disease-resistant locus is finally mapped between the markers SZC-7 and LSN-3-115 on A01, and between the markers ACMP00373 and SZC-37 on A08. The two disease-resistant loci show a dominant complementary genetic relationship. The present invention has found molecular markers closely linked to the disease-resistant genes contained in 'CR-20', completed the preliminary mapping of this locus, provided technical support for the future transfer of clubroot resistance in Brassica crops using the 'CR-20' material, and provided more options for the research on clubroot resistance breeding of Chinese cabbage and other Brassica crops in China.

Description

Technical Field

[0001] The present invention relates to the fields of crop breeding and biomolecular genetics, and particularly to the mapping of a pair of dominant complementary clubroot-resistant genes in Chinese cabbage, closely linked SSR molecular markers thereof, and their applications. Background Art

[0002] Clubroot, known as the "cancer of cruciferous plants", is a highly contagious soil-borne disease caused by the infection of Plasmodiophora brassicae. Plasmodiophora brassicae is an obligate parasite that infects the roots of living plants in the cruciferous family such as Brassica. When the root system is infected by Plasmodiophora brassicae, the parenchyma cells divide and enlarge, resulting in the formation of a large number of tumors in the plant root system, which blocks the physiological functions of the root system, causing the plant to grow slowly, wilt or die. In recent years, the main production areas of cruciferous vegetables and oil crops such as Chinese cabbage, cabbage, and rapeseed have been severely damaged by clubroot, and it shows a trend of large-scale spread, seriously restricting the planting and production of cruciferous crops.

[0003] Chinese cabbage (Brassica rapa ssp. pekinensis, A genome, 2n = 20) is an important leafy vegetable crop in the genus Brassica. However, at present, the damage of clubroot to Chinese cabbage is relatively serious. At present, the main methods for controlling clubroot include chemical, physical, biological, and disease-resistant breeding. Planting disease-resistant varieties is a safe, green and effective measure to control this disease. Therefore, screening and identifying disease-resistant germplasm resources, studying the genetic basis of clubroot resistance, mining excellent disease-resistant alleles, and analyzing their disease-resistant mechanisms are of great significance for promoting the cultivation of Chinese cabbage varieties resistant to clubroot.

[0004] Clubroot resistance (CR) genes are mostly derived from European turnips (Piao et al., 2007; 2009). The most widely used clubroot resistance materials are the ECD series of Brassica rapa subsp. rapa, especially ECD01, ECD02, ECD03, and ECD04 (Hasan et al., 2021). The CR loci of European turnips are mainly distributed on the A genome. So far, the identified CR loci from turnips include Crr1 (A08), Crr2 (A01), Crr3 (A03), Crr4 (A06), CRa (A03), CRb (A03), CRc (A02), CRk (A02), Rcr1 (A03), PbBa3.1 (A03), PbBa3.2 (A03), PbBa3.3 (A03), and PbBa8.1 (A08). Among them, the CRb gene is widely used in the breeding of disease-resistant Chinese cabbage varieties in China. However, clubroot CR genes have physiological race specificity. Due to the changes in physiological races and the action of selection pressure, disease-resistant varieties containing a single CR gene are prone to the problem of loss of resistance in production. Therefore, continuously exploring new disease-resistant genes is the key to the continuous, safe, and effective control of clubroot. Locating disease-resistant genes by constructing recombinant inbred lines and developing molecular markers linked to disease-resistant genes have great application value for cultivating disease-resistant varieties and shortening the breeding cycle. Summary of the Invention

[0005] The object of the present invention is to provide a pair of SSR molecular markers closely linked to the dominant complementary clubroot disease-resistant genes of Chinese cabbage and their applications, so as to solve the problems existing in the above-mentioned prior art. The genetic analysis and preliminary mapping of the disease-resistant genes contained in the resistant material 'CR-20' were carried out by using the genetic mapping method combined with the BSA sequencing method. Finally, the disease-resistant loci were mapped between the SZC-7 marker and the LSN-3-115 marker on chromosome 1 (A01) of Chinese cabbage, named BraPb1.3; between the ACMP00373 marker and the SZC-37 marker on chromosome 8 (A08), named BraPb8.4. There is a dominant complementary relationship between the two disease-resistant loci contained in 'CR-20'.

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

[0007] The present invention provides an SSR molecular marker closely linked to the clubroot disease-resistant gene of Chinese cabbage. The disease-resistant locus of the SSR molecular marker is located between the SZC-7 marker and the LSN-3-115 marker on chromosome 1 (A01) of Chinese cabbage, and between the ACMP00373 marker and the SZC-37 marker on chromosome 8 (A08);

[0008] The primer pairs for amplifying the SZC-7 marker are shown in SEQ ID NO: 1-2;

[0009] The primer pairs for amplifying the LSN-3-115 marker are shown in SEQ ID NO: 3-4;

[0010] The primer pairs for amplifying the ACMP00373 marker are shown in SEQ ID NO: 5-6;

[0011] The primer pairs for amplifying the SZC-37 marker are shown in SEQ ID NO: 7-8.

[0012] The present invention also provides a kit for identifying the clubroot resistance gene of Chinese cabbage, comprising primer pairs for amplifying the SZC-7 marker, LSN-3-115 marker, ACMP00373 marker and SZC-37 marker.

[0013] The present invention also provides the application of the SSR molecular marker or the kit in identifying the clubroot resistance gene of Chinese cabbage.

[0014] The present invention also provides the application of the SSR molecular marker or the kit in screening clubroot resistance materials of Chinese cabbage.

[0015] The present invention also provides the application of the SSR molecular marker or the kit in molecular marker-assisted breeding of Chinese cabbage.

[0016] The present invention also provides a method for identifying the clubroot resistance gene of Chinese cabbage, comprising the following steps:

[0017] Using the primer pairs for amplifying the SZC-7 marker, LSN-3-115 marker, ACMP00373 marker and SZC-37 marker to amplify the genomic DNA of the Chinese cabbage to be tested, analyzing the genotypes of the alleles amplified by the primer pairs, and determining whether the Chinese cabbage material to be tested has the clubroot resistance gene according to the genotype results.

[0018] Preferably, if both pairs of alleles amplified by the primer pairs are dominant, the Chinese cabbage material to be tested has the clubroot resistance gene.

[0019] Preferably, the reaction system for amplification is: 10 μL of amplification premix, 2 μL of DNA template, 1 μL each of the upstream primer and the downstream primer, and 6 μL of ddH2O.

[0020] Preferably, the reaction program for amplification is: 94 °C for 7 min; 94 °C for 2 min; 55 °C for 30 s, 72 °C for 30 s, 72 °C for 5 min; hold at 4 °C.

[0021] The present invention discloses the following technical effects:

[0022] It was found through experiments in the present invention that the parent 'CR-20' showed disease resistance to the clubroot fungus SXTB104, and the parent 'BJN3-1' showed susceptibility to the clubroot fungus SXTB104. The hybrid offspring F1 showed disease resistance after inoculation with SXTB104. Therefore, the disease-resistant gene contained in the resistant parent material 'CR-20' is dominant. The results after inoculating the F2 population with SXTB104 showed that the ratio of the number of disease-resistant to susceptible individual plants was 9:7. Therefore, the disease-resistant trait is controlled by two pairs of alleles, and the material shows resistance only when both pairs of genes are dominant, proving that there is a dominant complementary relationship between the two loci.

[0023] The disease-resistant gene contained in the resistant material 'CR-20' was genetically analyzed and preliminarily mapped by combining the genetic map mapping method with the BSA sequencing method. Finally, the disease-resistant locus was mapped between the A01: SZC-7 marker and the LSN-3-115 marker, named BraPb1.3; between the A08: ACMP00373 marker and the SZC-37 marker, named BraPb8.4. Then through F 2:3 The association analysis of the population phenotype and genotype confirmed the dominant complementary relationship between the two disease-resistant loci contained in 'CR-20'. The present invention completed the preliminary mapping of the disease-resistant loci of 'CR-20' and developed molecular markers closely linked to the two loci. The two-locus linked markers can be directly used for the transfer of the clubroot disease-resistant gene of 'CR-20'. The present invention provides technical support for the future work of transferring the clubroot disease-resistant gene using the 'CR-20' material, and provides more options for the research on clubroot disease resistance breeding of Chinese cabbage and other Brassica crops in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 It is the research technical roadmap of the molecular marker of the present invention;

[0026] Figure 2 It is the identification result of the parental disease resistance; 1 is the susceptible parent BJN3-1, and 2 is the disease-resistant parent CR-20;

[0027] Figure 3 It is the identification result of the F1 disease resistance;

[0028] Figure 4Verification results of CRa disease-resistant gene; in the left half, 0 represents 5000bp Marker, 1 represents 222 (CRa), 2 represents BJN3-1, and 3 represents CR-20; in the right half is the secondary verification, with the same order as the left half;

[0029] Figure 5 Verification results of CRb and CRd disease-resistant genes; A is the verification result of CRb, with linked markers TCR01 and TCR05 (Piao et al, 2004), and DNA from left to right: 1 represents BJN3-1, 2 represents 222 (CRb), 3 represents CR-20; B is the verification result of CRd, with linked markers yau376 and yau106 (Fu Pengyu, 2019), and DNA from left to right: 1 represents BJN3-1, 2 represents 85-74 (CRd), 3 represents CR-20;

[0030] Figure 6 F2 disease resistance identification results; from left to right are CK, level 0, level 1, level 2, and level 3;

[0031] Figure 7 BSA sequencing trait association interval mapping diagram;

[0032] Figure 8 Genetic map of chromosome A08;

[0033] Figure 9 Genetic map of chromosomes A01 - A03;

[0034] Figure 10 Genetic map of chromosomes A04 - A06;

[0035] Figure 11 Genetic map of chromosomes A07, A09, and A10;

[0036] Figure 12 For F 2:3 Analysis results of the disease index of F families;

[0037] Figure 13 Distribution of disease-resistant loci obtained by composite interval mapping; A01 represents chromosome 1, and A08 represents chromosome 8.

[0038] Figure 14 Electrophoresis diagram of amplification products of tightly linked markers SZC-7 and LSN-3-115 for disease-resistant genes located on chromosome A01 between resistant and susceptible parents; P1 is the susceptible parent BJN3-1, P2 is the resistant parent CR-20, and F2 represents individual plants from part of the F2 population;

[0039] Figure 15Electrophoresis diagram of amplification products of tightly linked markers SZC37 and ACMP00373 for disease-resistant genes located on chromosome A08 between resistant and susceptible parents; P1 is the susceptible parent BJN3-1; P2 is the resistant parent CR-20; F2 represents individual plants of a partial F2 population. Detailed implementation mode

[0040] The various exemplary implementation modes of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing specific implementation modes and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0043] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation modes of the present invention specification, which are obvious to those skilled in the art. Other implementation modes obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0044] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0045] Example 1

[0046] 1. Test materials

[0047] The selected test materials are all from the Vegetable Molecular Laboratory of the College of Horticulture, Shenyang Agricultural University. The disease-resistant material 'CR-20' is selected as the male parent, and the female parent material is the disease-susceptible material 'BJN3-1'.

[0048] Test strains: The root-knot bacteria 'SXTB104' was selected as the test strain. The bacterium was identified by the SCD identification method (Pang et al., 2020), and the results showed that the bacterium belonged to the Pb8 physiological race (currently stored in the strain storage refrigerator of the Vegetable Molecular Laboratory of Shenyang Agricultural University).

[0049] 2. Test methods

[0050] The hybridization of the disease-resistant material 'CR-20' and the susceptible material 'BJN3-1' and multi-generation self-pollination were used to construct F 2 groups and F 2:3 The disease resistance gene contained in Chinese cabbage 'CR-20' was preliminarily located by using BSA sequencing combined with traditional genetic map positioning method. Figure 1 .

[0051] 2.1 Method of inoculation of root knot fungus

[0052] The Pb8 physiological race 'SXTB104' was selected as the test strain for later inoculation and identification. This strain will not cause disease in 'CR-20', but 'BJN3-1' showed susceptibility to the disease when inoculated with this strain. The inoculation method is the bacterial liquid injection method. The results of many years of experiments in this laboratory show that this method is not only simple and convenient to operate, but also has relatively accurate inoculation and identification results. The specific inoculation experimental method is as follows:

[0053] 2.1.1 Preparation of bacterial solution

[0054] 1) Take out 50 g of SXTB104 bacterial blocks from the special storage refrigerator for bacterial strains and thaw them at room temperature. After the bacterial blocks are thawed, put them into a wall breaking machine and add 200 mL of sterile water to fully grind them. Filter the mixed liquid with 8 layers of medical sterile gauze, and pour the filtered bacterial liquid into a sterilized volumetric flask.

[0055] 2) Use a pipette to draw the bacterial solution into a 50 mL centrifuge tube, place it in a centrifuge and balance it. Set the program to centrifuge at 4°C and 1000g for 10 minutes. Discard the supernatant and add sterile water. Repeat 3 times.

[0056] 3) Mix the treated bacterial solution thoroughly on a vortex shaker, pipette 100 μL of the bacterial solution into a 1.5 mL centrifuge tube, then add 900 μL of sterile water and shake up and down for 30 seconds. Pipette 100 μL of the diluted bacterial solution into a 1.5 mL centrifuge tube, add 200 μL of aniline blue solution and 700 μL of sterile water, mix well, and stain for 5 minutes. After staining, use a hemocytometer to measure the concentration of the bacterial solution and determine the concentration of the bacterial solution at 1×10 7 pc·mL -1 , and then store in a 4°C refrigerator. When conducting inoculation tests, try to grind the samples on the same day they are used to avoid long-term storage.

[0057] 2.1.2 Inoculation with Plasmodiophora brassicae

[0058] (1) In March 2021, the parents, F1 generation, and 229 F2 generations and F 2:3 families obtained through self-pollination were sown in sterilized 72-well trays, placed in the No. 3 greenhouse of Shenyang Agricultural University (20℃ - 25℃), and watered and pest-controlled on time. When the plants had two leaves and one heart, about 20 days later, inoculation was carried out using the mycelium injection method.

[0059] (2) Prepare sterilized 5 mL syringes and beakers, and inoculate according to the standard of 2 mL of mycelium solution per plant. Water the plants again the day after inoculation. The photoperiod is 16 h light / 8 h dark. Pay attention to observing the growth status of the plants every day to ensure that the soil remains moist.

[0060] (3) Phenotypic identification: Disease resistance identification was carried out 45 days after inoculation. Adjust the identification time appropriately according to the disease incidence of the susceptible parent material. When conducting the inoculation result identification, the soil humidity can be appropriately reduced to ensure the accuracy of the disease incidence results. Wash the excess substrate from the roots of the plants with clean water, and try to keep the roots intact and clearly visible during the washing process. Observe the disease incidence and conduct grading statistics.

[0061] 2.2 Disease severity level identification

[0062] In May 2021, seven weeks after inoculation, the disease resistance of the test materials was investigated. Since the disease-causing part of Plasmodiophora brassicae is in the roots of the plants, when conducting the disease condition identification, gently pull out the plants in the trays and wash them gently with water. The treated plants were graded and statistically analyzed according to the grading method proposed by Buczacki et al. in 1975. The specific standards of this grading method are shown in Table 1 below:

[0063] Table 1 Grading standards for clubroot disease

[0064]

[0065] 2.3 Population construction

[0066] Hybridize the disease-resistant material 'CR-20' with the susceptible material 'BJN3-1' to obtain the F1 population. Sow the harvested F1 population in 72-well trays. After natural vernalization, randomly select 5 healthy plants and transplant them to the experimental base in the No. 3 greenhouse of the Horticulture College of Shenyang Agricultural University. Randomly select 200 seeds from the harvested F2 population through artificial pollination and place them in a sterilized petri dish. Add sterile water to the petri dish until the filter paper in the petri dish is completely wet. Conduct dark treatment. After the seeds grow into small buds, transfer them to a special vernalization refrigerator in the laboratory, and control the temperature at 4℃ (water regularly, with a light / dark cycle of 18 h / 6 h).

[0067] The vernalized seedlings were sown in 72-well trays. When they grew to an appropriate height, they were transplanted to the experimental base in Shed No. 3 of the Horticulture College of Shenyang Agricultural University, and F 2:3 families were obtained through artificial pollination.

[0068] 2.4 DNA Extraction and Concentration Detection

[0069] In this experiment, the method for extracting leaf DNA was the modified CTAB method. The DNA extracted by this method has the advantages of high concentration and long preservation time. The specific experimental steps are as follows:

[0070] I. Pre-cool anhydrous ethanol and 70% ethanol in a -20°C refrigerator in advance. Take fresh leaves about 1 cm in length and width and put them into a 2 mL centrifuge tube. Add sterilized steel beads. The plant numbers and centrifuge tube numbers are arranged in one-to-one correspondence on the operation board. Wear anti-freezing gloves and transfer the samples to a foam box filled with liquid nitrogen for freezing for 5 minutes. Then take out the operation board and place it horizontally in a vibrating grinder for grinding for 5 minutes. Depending on the freezing effect and the sample volume, perform secondary vibrating grinding as appropriate.

[0071] II. Place the ground samples on the experimental bench and wait for them to reach room temperature. Use a pipette to add 600 μL of 2% CTAB extraction buffer that has been preheated in a 65°C water bath for 40 minutes. To ensure the uniformity of the mixture, invert the operation board up and down after adding the CTAB extraction buffer.

[0072] III. Fix the mixed samples on the floats in sequence and place them in a 65°C water bath for 45 minutes. During this period, take out the floats and shake them once every 10 minutes to ensure that the samples are evenly heated.

[0073] IV. Prepare chloroform and isopentyl alcohol, wear a gas mask and prepare chloroform according to a volume ratio of 24:1 in a fume hood. After the water bath is completed, insert the samples into the operation board in sequence, and avoid cross-contamination during the process of opening the lid. Add 600 μL of chloroform to them, shake them up and down for 20 s, and then transfer them to a centrifuge for centrifugation for 15 minutes, with the program of 12000 r / min.

[0074] V. After centrifugation, use a 1000 μL pipette to aspirate 450 μL of the supernatant into a 1.5 mL centrifuge tube. During the aspiration process, different specifications of centrifuge tubes correspond to each other according to the numbers, change the pipette tips in time and avoid inhaling impurities. After the supernatant aspiration is completed, add 900 μL of pre-cooled anhydrous ethanol. After checking for no leakage or omission, place the samples horizontally in a -20°C refrigerator and let them stand for more than 40 minutes. After standing, transfer them to a centrifuge with a program of 12000 r / min for centrifugation for 10 minutes.

[0075] VI. After centrifugation, take out the sample, discard the supernatant in the centrifuge tube, and then add 600 μL of pre-cooled 70% ethanol with a 1000 μL pipette. After adding the drugs, centrifuge for 5 min in a centrifuge with a program of 12000 r / min.

[0076] VII. After centrifugation, discard the supernatant in the centrifuge tube. Place the centrifuge tube containing DNA horizontally in the fume hood and air dry it for 24 h. After ventilation, add 200 μL of ultrapure water and dissolve it at room temperature for 2 hours. Then detect the DNA concentration by methods such as enzyme-linked immunosorbent assay or ultraviolet spectrophotometry.

[0077] 2.5 Screening of polymorphic markers

[0078] After verifying that the extracted DNA meets the standards for further experiments, use polyacrylamide gel electrophoresis combined with SSR markers and Indel markers developed based on the Chinese cabbage genome (Ramchiary et al., 2011; Jiao Yang, 2022) to screen primers with polymorphism between parents. The selected primers should cover 10 chromosomes and be evenly distributed on each chromosome, so as to use the primers with polymorphism to identify the genotypes of F2 generation individual plants and construct a genetic map. The synthesis of all primers was completed by Suzhou Hongxun Company. All experimental steps for screening polymorphic markers are as follows:

[0079] In this invention, the PCR amplification adopts a 20 μL system, and the addition amounts of each drug and the reaction program are shown in Table 2 below:

[0080] Table 2 PCR amplification system

[0081]

[0082] The PCR reaction program is shown in Table 3:

[0083] Table 3 PCR amplification program

[0084]

[0085] The amplified products were detected by polyacrylamide gel electrophoresis.

[0086] 3. BSA sequencing and QTL analysis

[0087] 3.1 BSA sequencing

[0088] According to the clubroot identification results of the F2 generation population, 40 extremely resistant and 40 extremely susceptible individual plants were selected from 229 F2 populations to construct a disease-resistant mixed pool (R pool) and a susceptible mixed pool (S pool). The DNA of the two mixed pools and the parents was extracted respectively, and the BSA sequencing work was completed by Shanghai Personal Biotechnology Co., Ltd. The main steps of genomic BSA sequencing are as follows:

[0089] i. Total genomic DNA extraction;

[0090] ii. Sequencing library preparation;

[0091] iii. High-throughput sequencing on the machine;

[0092] iv. Data quality control;

[0093] v. Alignment with the reference genome;

[0094] vi. SNP and Indel detection and annotation;

[0095] vii. Target trait mapping;

[0096] viii. Target trait region analysis.

[0097] 3.2 Construction of genetic map

[0098] After screening and counting the primers with different banding patterns between the parents, further verify whether they are still polymorphic in the F2 population and record the genotypes. Those consistent with the genotype of the susceptible parent 'BJN3-1' are recorded as 'a', those consistent with the genotype of the resistant parent 'CR-20' are recorded as 'b', and the heterozygous bands are recorded as 'h'. Use the software joinmap 4.0 to construct the genetic map, set the LOD value to 2.0, select kosambi's as the mapping function, verify whether the selected markers are linked, and the constructed genetic map should cover 10 chromosomes and be evenly distributed on each chromosome.

[0099] 3.3 QTL analysis

[0100] After the map construction is completed, combined with the disease incidence identified in F 2:3 Use the formula: disease index = ∑(number of diseased plants at each level × corresponding level) / total number of plants in the surveyed population × highest level value to calculate the disease index. Those with a disease index ≥ 25 are recorded as susceptible, and those with a disease index less than 25 are recorded as resistant. Infer the F2 phenotype based on the calculation results and use the software QTLIciMapping for QTL analysis.

[0101] 4. Results and analysis

[0102] 4.1 Genetic analysis

[0103] 4.1.1 Resistance identification of parents and F1

[0104] Inoculation identification was carried out on the two parents 'CR-20' and 'BJN3-1', as well as F1, and the results are as Figures 2 - 3As shown, the results showed that 36 'CR-20' plants were resistant to the disease, 36 'BJN3-1' plants were susceptible to the disease, and all 30 F1 plants were resistant to the disease. These results indicated that the disease-resistant trait exhibited by 'CR-20' was controlled by dominant genes.

[0105] To further verify whether 'CR-20' contained known disease-resistant genes, DNA fragments of materials containing the disease-resistant gene and DNA fragments of 'BJN3-1' were amplified using the existing CRa, CRb, and CRd linked markers in the laboratory. For the verification of CRa and CRb, the disease-resistant material '222' was selected, and for the verification of CRd, the disease-resistant material '85-74' was selected. CRa was verified by agarose gel electrophoresis, and CRb and CRd were verified by polyacrylamide gel electrophoresis. As Figures 4 - 5 shown, the results showed that 'CR-20' did not contain the above-mentioned known disease-resistant gene fragments.

[0106] 4.1.2 Resistance identification of the F2 population

[0107] The results of inoculating 229 F2 plants showed that the ratio of resistant to susceptible plants was 138:91. A chi-square test was performed with the theoretical value of 9:7 (see Table 4), and the result was x 2 = 1.28 < x 2 (0.05), showing no significant difference from the theoretical value and conforming to the 9:7 segregation ratio. As Figure 6 shown, the results showed that the resistance of 'CR-20' to Plasmodiophora brassicae was controlled by two genes together, and resistance was only exhibited when both genes were dominant.

[0108] Table 4 Chi-square test of F2

[0109]

[0110] 4.2 BSA sequencing data analysis

[0111] 4.2.1 GPS identification of genomic regions

[0112] GradePool-Seq is a new quantitative trait gene mapping method developed by the Han Bin research group. This research proposed a quantitative trait locus (QTL) mapping method, namely GradedPool-Seq (GPS) for rapid mapping of QTLs. It analyzes the genomic sequencing of gradient pool samples of F2 offspring and then performs BSA on the machine. Theoretically, the lower the P value, the higher the correlation between the variation and the phenotype. However, due to a large amount of background noise in SNP / Indel signals in distant genetic crosses, it is necessary to consider reducing the background noise. The noise reduction algorithm implemented in the research is a non-overlapping sliding window method, that is, within a determined genomic interval (about 400 kb), calculate the ratio of the number of variations significantly exceeding the threshold to the total number of variations. The interval with the largest ratio is the interval where the proportion of SNPs / Indels groups with significant p values is the highest. After adopting the strategy of reducing background noise, the results are presented in the form of a ratio map. The areas with high ratios are the trait association intervals, see Figure 7 . The candidate interval is located at 9.8 Mb - 12.2 Mb on chromosome A08 and is named BraPb8.4 (Table 5).

[0113] Table 5 BSA candidate intervals

[0114]

[0115] 4.3 Construction of the F2 genetic map

[0116] After genetic analysis of 'CR-20' and 'BJN3-1' and the population constructed by them, the present invention concluded that the disease-resistant trait is controlled by two dominant complementary genes, while BSA only detected signals at one locus. Therefore, the present invention constructed a genetic map for this material and used QTL mapping methods to determine the disease-resistant loci on other chromosomes.

[0117] 4.3.1 Screening of polymorphic markers between parents

[0118] SSR markers and Indel markers developed based on the Chinese cabbage genome were used to screen for polymorphic markers between parents, and markers with clear bands, obvious polymorphism, and co-dominance were selected for the analysis of the F2 population and subsequent experiments.

[0119] 4.3.2 Marker verification and map construction of the F2 population

[0120] The BSA test results identified a disease-resistant locus located at 9.8 Mb - 12.2 Mb on chromosome A08. Therefore, the markers on chromosome A08 were analyzed first, and the selected markers on chromosome A08 were used for the verification of the F2 population. Among them, a total of 13 pairs of SSR markers and 2 pairs of Indel markers were used to construct a genetic map, and the software Joinmap 4.0 was used for analysis. The LOD threshold was set to ≥2.5, and the kosambi algorithm was adopted. The analysis results showed that 5 pairs of markers failed to link. Subsequently, SSR primers were designed using the software SSR hunter according to the candidate interval given by BSA to increase the number of linked markers in the candidate interval. One pair of primers was designed every 30 Wbp, and a total of 30 pairs of primers were designed. These markers were screened, and the qualified markers were added to the genetic map. The final genetic map obtained is as Figure 8 shown. The length of this genetic map is 42.3 cM, the number of markers is 16 pairs, including one pair of Indel markers, the average map distance is 2.64 cM, and the density is 0.38 per cM.

[0121] After completing the genetic map on chromosome A08, primers with polymorphisms between parents on the remaining chromosomes were continuously screened to ensure that there were 10 linked markers in each map as much as possible. After the genetic maps on the remaining chromosomes were constructed using the same method, they were named in the order of A01 - A10. The genetic maps on the remaining part of the chromosomes are as Figures 9 - 11 shown:

[0122] The constructed genetic maps were analyzed as a whole, and the results are shown in Table 6. The results showed that the total length of this map is 564.5 cM, the average map distance is 5.82 cM, and the density is 0.17 cM. Among them, the molecular marker density on chromosome A08 is the largest, which is 0.38 per cM, and the molecular marker density on chromosome A10 is the smallest, which is 0.08 per cM; from the perspective of the average map distance, the distance on chromosome A10 is the largest, which is 12.7 cM, and the distance on chromosome A08 is the smallest, which is 2.64 cM; generally speaking, the markers on chromosome A10 are the most evenly distributed, and the genetic map distance on chromosome A03 is the longest, which is 73.2 cM.

[0123] Table 6 Analysis of genetic linkage map

[0124]

[0125]

[0126] 4.4 QTL analysis results

[0127] 4.4.1 F 2:3 Disease index calculation

[0128] After counting F 2:3After investigating the family incidence, using the formula: ∑(number of diseased plants at each level × corresponding level) / total number of plants in the surveyed population × highest level value; calculate F 2:3 family disease index and analyze the results. As shown in Table 7, the results show that F 2：3 In the population, the number ratio of resistant to susceptible is 116:76. The chi-square test result is x 2 = 1.19 < x 2 (0.05), showing no significant difference from the theoretical value of 9:7 and still conforming to the 9:7 segregation ratio. This result further confirms that the disease resistance of 'CR-20' is controlled by two pairs of dominant complementary genes.

[0129] Table 7 F 2:3 Chi-square test results

[0130]

[0131] Enter the calculated disease index into SPSS software to analyze the peak value. The results are as Figure 12 shown. The analysis results show that the disease index of the F 2:3 family is skewed normal distribution, meeting the basic characteristics of quantitative traits and meeting the basic requirements for further QTL experiments.

[0132] 4.4.2 QTL analysis

[0133] Combine the calculated disease index of the F 2:3 family with the constructed genetic map, and use the software QTL IciMapping to analyze the QTL results. As Figure 13 and Table 8 show, finally lock two disease resistance loci, BraPb1.3 and BraPb8.4, between 6.3-7.4 Mb on chromosome A01, between the SZC-7 marker and the LSN-3-115 marker; in the interval of 9.9-10.5 Mb on chromosome A08, between the ACMP00373 marker and the SZC-37 marker. The LOD values are 2.54 and 11.44 respectively, and the genetic contribution rates are 4.42% and 23.95% respectively. (The Chinese cabbage reference genome is Brara Chiifu V 1.5 genome, see www.brassicadb.cn) The electrophoresis maps of the four linked markers in the resistant and susceptible materials and F2 single plants are shown in Figure 14 and Figure 15 , and the genotype consistent with the resistant parent 'CR-20' band is the resistant genotype, the genotype consistent with the susceptible parent 'BJN3-1' band is the susceptible genotype, and the one with both bands is the heterozygous genotype.

[0134] Table 8 QTL analysis of CR-20 disease resistance loci

[0135]

[0136] 4.4.3F 2:3 Verification of the Association Analysis between Population Phenotype and Genotype

[0137] To further verify that the disease-resistant trait is controlled by two loci, the association analysis was performed on the F 2:3 family phenotype and F2 genotype. The genotypes of 4 linked markers were counted. Those consistent with the genotype of the susceptible parent were denoted as A, and those consistent with the genotype of the disease-resistant parent were denoted as B. The families containing the genotype of the susceptible parent were grouped into one category, denoted as category A, and the families without the genotype of the susceptible parent were grouped into one category, denoted as category B. The average disease index of the two parts was calculated respectively. The calculation results are shown in Table 9 and Table 10:

[0138] Table 9 Analysis of the Average Disease Index of Category B

[0139]

[0140]

[0141] Table 10 Analysis of the Average Disease Index of Category A

[0142]

[0143] Note: "-" represents genotype deletion.

[0144] Analyzing the calculation results, the average disease index of category A is 40.76, which is higher than 25 and belongs to the highly susceptible type. The average disease index of category B is 9.57, which belongs to the highly resistant type. The ratio of the number of disease-resistant to susceptible families in the two parts is 111:81. The chi-square test result is x 2 =0.13 < x 2 (0.05), showing no significant difference from the theoretical value. This result further proves that the disease-resistant trait of 'CR-20' is controlled by two genes, and only when both alleles are dominant can this material show resistance, further clarifying the dominant interaction relationship between the two loci. In addition, the linked markers developed by the present invention based on the two loci can be directly used for the transfer of the clubroot resistance locus of the disease-resistant material 'CR-20'.

[0145] In summary, the present invention has completed the genetic analysis and preliminary mapping of the disease-resistant locus of 'CR-20', and developed molecular markers tightly linked to the two loci. The two-locus linked markers can be directly used for the transfer of clubroot resistance genes in other Brassica crops.

[0146] Specific information of the literature involved in the above embodiments:

[0147] (1) Pang W, Liang Y, Zhan Z, Li X, Piao Z. Development of a sinitic clubroot differential set for the pathotype classification of Plasmodiophorabrassicae. Front Plant Sci, 2020, 11, 568771.

[0148] (2) Piao ZY, Deng YQ, Choi SR, Park YJ, Lim YP. SCAR and CAPS mapping of CRb, a gene conferring resistance to Plasmodiophora brassicae in Chinese cabbage (Brassica rapa ssp. pekinensis). Theor Appl Genet, 2004, 108, 1458 - 1465.

[0149] (3) Fu Pengyu, Mapping, Cloning and Functional Identification of the Clubroot Resistance Gene CRd in Chinese Cabbage

D

[0150] (4) Jiao Yang, Gene mapping of clubroot resistance based on Chinese cabbage "CR - 096"

D

[0151] (5) Ramchiary N, Nguyen VD, Li X, Hong C, Dhandapani V, Choi SR, Yu G, Piao ZY, Lim YP. Genic Microsatellite Markers in Brassica rapa: Development, Characterization, Mapping, and Their Utility in Other Cultivated and Wild Brassica Relatives. DNA Research, 2011, 18, 305 - 320.

[0152] The above - described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a reagent for detecting SSR molecular markers in identifying Chinese cabbage clubroot resistance genes, screening Chinese cabbage clubroot resistance materials, or Chinese cabbage clubroot resistance auxiliary breeding, characterized in that: The disease resistance loci of the SSR molecular marker are located between the SZC-7 marker and the LSN-3-115 marker on chromosome 1 of Chinese cabbage, and between the ACMP00373 marker and the SZC-37 marker on chromosome 8; The primer pair used to amplify the SZC-7 marker is shown in SEQ ID NO: 1-2; The primer pair used to amplify the LSN-3-115 marker is shown in SEQ ID NO: 3-4; The primer pair used to amplify the ACMP00373 marker is shown in SEQ ID NO: 5-6; The primer pair used to amplify the SZC-37 marker is shown in SEQ ID NO: 7-8; The Chinese cabbage reference genome is the Brara Chiifu V 1.5 genome.

2. The use of a kit for identifying Chinese cabbage clubroot resistance genes in identifying Chinese cabbage clubroot resistance genes, screening Chinese cabbage clubroot resistance materials or Chinese cabbage clubroot resistance auxiliary breeding, characterized in that: Includes primer pairs for amplifying SZC-7 marker, LSN-3-115 marker, ACMP00373 marker, and SZC-37 marker; The primer pair used to amplify the SZC-7 marker is shown in SEQ ID NO: 1-2; The primer pair used to amplify the LSN-3-115 marker is shown in SEQ ID NO: 3-4; The primer pair used to amplify the ACMP00373 marker is shown in SEQ ID NO: 5-6; The primer pair used to amplify the SZC-37 marker is shown in SEQ ID NO: 7-8.

3. A method for identifying a Chinese cabbage clubroot resistance gene, characterized in that: The following steps are involved: The genomic DNA of the Chinese cabbage to be tested is amplified using the primer pair for amplifying the SZC-7 marker, the LSN-3-115 marker, the ACMP00373 marker and the SZC-37 marker in claim 1, the genotype of the allele amplified by the primer pair is analyzed, and whether the Chinese cabbage material to be tested has a clubroot resistance gene is determined according to the genotype result.

4. The method according to claim 3, characterized in that If both pairs of alleles amplified by the primer pair are dominant, the Chinese cabbage material to be tested has a clubroot resistance gene.

5. The method according to claim 3, characterized in that The amplification reaction system is: 10 μL of amplification premix, 2 μL of DNA template, 1 μL of upstream primer and downstream primer respectively, and 6 μL of ddH2O.

6. The method according to claim 3, characterized in that The amplification reaction procedure is: 94°C for 7 min; 94°C for 2 min; 55°C for 30 s, 72°C for 30 s, 72°C for 5 min; and hold at 4°C.

Images