Molecular marker for radish clubroot resistance breeding and construction method thereof

Abstract

The invention discloses a molecular marker for radish clubroot resistance breeding and a construction method, and particularly relates to an InDel molecular marker developed based on a radish clubroot resistance gene locus RsCr7. The establishment of the InDel molecule provides important technical support for rapid creation of radish disease-resistant materials and efficient cultivation of multi-resistance varieties, not only enriches radish clubroot resistance gene resources, but also provides a new molecular tool for disease-resistant breeding.

Description

Technical Field

[0001] This application belongs to the fields of plant genetics and plant pathology, and specifically relates to a molecular marker for breeding resistance to clubroot disease in radishes and its construction method. Background Technology

[0002] Clubroot is a soil-borne disease that seriously damages cruciferous crops, causing severe yield losses worldwide. Due to the widespread lack of effective resistance genes in Chinese radish germplasm, clubroot has become a serious threat to the sustainable development of my country's radish industry.

[0003] In recent years, some progress has been made in the research of radish resistance genes, such as the identification of two dominant single-gene resistance loci, Crs1 and RsCr6, and five QTLs (RsCr1 to RsCr5) controlled by recessive polygenic genes. Furthermore, researchers have developed molecular markers linked to the resistance loci RsCr2 and RsCr6 and successfully applied them to the breeding of disease-resistant varieties. However, most of the resistance genes discovered so far are physiological race-specific, and long-term cultivation of varieties with a single resistance gene may lead to the evolution of pathogen races, reducing varietal resistance.

[0004] Therefore, in order to improve the durability and stability of disease-resistant varieties, it is the current research focus and development direction to discover new resistance gene loci and to aggregate multiple resistance genes into the same material through molecular marker-assisted selection. Summary of the Invention

[0005] The purpose of this application is to develop a novel InDel molecular marker by studying the RsCr7 gene locus for resistance to clubroot disease in radishes, so as to apply it to the breeding of clubroot-resistant varieties of radishes.

[0006] To achieve the above objectives, this application provides the following technical solution:

[0007] A molecular marker for breeding radish with resistance to clubroot disease, the molecular marker comprising: forward primer: 5'-TATGTTAGTTTCTGCGGCAATAGG-3'; (SEQ ID NO.1), and reverse primer: 5'-GAAGCTAGGGAAGACTTGGTGT-3' (SEQ ID NO.2).

[0008] This application also provides a method for constructing molecular markers for clubroot disease resistance breeding in radishes. The method includes: initial mapping of the clubroot disease resistance gene locus RsCr7 in radishes; fine mapping of the initially mapped clubroot disease resistance gene locus RsCr7 in radishes; cloning of the candidate clubroot disease resistance gene RsCr7 (SEQ ID NO.3) and allele Rscr7 (SEQ ID NO.4) selected by fine mapping; and then developing markers based on the sequence differences between alleles to obtain molecular markers for clubroot disease resistance breeding in radishes.

[0009] Optionally, the initial localization of the radish clubroot resistance gene locus RsCr7 includes: parental selection and population construction; inoculation experiments and disease resistance assessment of the selected parents and constructed populations; genomic DNA extraction, pool construction, and whole-genome sequencing analysis of the parents and populations after disease resistance assessment; comparison of the genome sequencing data with the reference genome to obtain SNPs for each sample, calculation of the SNP-index of the resistant pool and the susceptible pool, and the difference between the two, ΔSNP-index, to achieve initial localization.

[0010] Optionally, the fine mapping of the radish clubroot resistance gene locus RsCr7 after initial mapping includes: screening InDel loci; designing primers and performing PCR verification on the screened InDel loci; performing InDel molecular marker typing on individual plants in the F2 population; and then conducting genetic linkage analysis in combination with phenotypic data to draw a linkage map to achieve fine mapping of RsCr7.

[0011] Optionally, the marker development for the finely mapped radish clubroot resistance gene locus RsCr7 includes: performing bioinformatics analysis on genes within the finely mapped region, obtaining candidate genes for the RsCr7 locus based on gene annotation information; cloning alleles of resistant and susceptible materials based on the candidate gene information, and developing molecular markers based on allele sequence differences to obtain molecular markers for radish clubroot resistance breeding.

[0012] This application also provides an application of a molecular marker for breeding radish resistance to clubroot disease, wherein the molecular marker is used for breeding disease-resistant varieties.

[0013] Compared with the prior art, the beneficial effects of this application are as follows:

[0014] The molecular markers developed in this application can accurately distinguish between disease-resistant individuals carrying the RsCr7 gene and disease-susceptible individuals not carrying the gene, thus providing an effective means for breeding disease-resistant varieties. Attached Figure Description

[0015] Figure 1This is a flowchart illustrating a method for constructing molecular markers for clubroot disease resistance breeding in radishes, provided in one embodiment of this application.

[0016] Figure 2 This is a schematic diagram of the location results of the RsCr7BSA-seq of the radish clubroot resistance gene locus;

[0017] Figure 3 This is a schematic diagram showing the fine mapping results of the RsCr7 gene locus, which is the resistance gene to clubroot disease in radishes.

[0018] Figure 4 This is a schematic diagram showing the sequence alignment results of RsCr7, a candidate gene for resistance to clubroot disease in radishes, and its allele Rscr7.

[0019] Figure 5 This is a schematic diagram illustrating the verification of InDel marker gene typing and resistance identification;

[0020] Figure 6 A schematic diagram showing the results of indoor inoculation identification of resistance to clubroot disease in *Clerodendrum chinense*.

[0021] Figure 7 A schematic diagram showing the results of indoor inoculation identification of clubroot resistance of C35019, a resistance-modified material for the spring-growing plant;

[0022] Figure 8 A schematic diagram showing the results of indoor inoculation identification of clubroot resistance in the white roundflower A / C35019;

[0023] Figure 9 This is a schematic diagram showing the results of field natural disease resistance identification of white round flower A / C35019 and spring old clubroot resistance. Detailed Implementation

[0024] Specific embodiments of this application will now be described in detail with reference to the accompanying drawings. While specific embodiments of this application are shown in the drawings, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of this application and to fully convey the scope of this application to those skilled in the art.

[0025] To facilitate understanding of the embodiments of this application, the following will provide further explanation and description with reference to the accompanying drawings and specific embodiments, and the accompanying drawings do not constitute a limitation on the embodiments of this application.

[0026] In one embodiment, this application provides a molecular marker for breeding radish resistance to clubroot disease, the molecular marker comprising:

[0027] Forward primer: 5'-TATGTTAGTTTCTGCGGCAATAGG-3'; (SEQ ID NO.1)

[0028] Reverse primer: 5'-GAAGCTAGGGAAGACTTGGTGT-3'. (SEQ ID NO.2)

[0029] In another embodiment, such as Figure 1 As shown, this application provides a method for constructing molecular markers for clubroot resistance breeding in radishes, comprising the following steps:

[0030] S100: Preliminary localization of the radish clubroot resistance gene RsCr7, specifically including the following steps:

[0031] S101: Parental selection and population building;

[0032] In this step, this application selected the immune-grade resistant material 'Snow White' (identified as immune to the physiological race Pb3 of *Plasmodium*, with its resistance controlled by a pair of dominant genes) and the susceptible material 'Sand Pot Radish' as ​​parents. The F1 generation was obtained through hybridization, and the F2 population was obtained through self-pollination of the F1 generation for subsequent gene mapping and marker verification. Specifically, the selected materials are shown in Table 1.

[0033] Table 1

[0034]

[0035] In Table 1, Sn indicates that the tested material is an inbred line, and n indicates the number of inbred generations; C indicates that the tested material is a conventional variety; BCn indicates that the tested material is a backcross population of sterile lines, and n indicates the number of backcross generations with the maintainer line.

[0036] S102: Conduct inoculation experiments and disease resistance assessments on the selected parents and the constructed population;

[0037] In this step, the parent plants, F1 and F2 populations were subjected to microspore inoculation experiments with *Plasmodiophora* to assess their disease resistance. Based on the disease incidence, the F2 population was divided into two groups: extremely resistant (grade 0) and extremely susceptible (grade 4).

[0038] Specifically, the inoculation concentration of *Plasmodiophora stearothermiae* microspores was 1 × 10⁻⁶. 8 ml -1 Each plant was inoculated with 5 ml of the solution and placed in a light culture room with a daytime temperature of 25℃~30℃, a nighttime temperature of 15℃~20℃, and a photoperiod of 12h / 12h. Disease incidence was assessed 55 days after inoculation. Based on the disease incidence, this application categorizes disease incidence into the following five levels:

[0039] Grade 0: Radish root system is growing normally, with no tumors;

[0040] Grade 1: The root system has a few small tumors on its fibrous roots or lateral roots, and the main root is not diseased.

[0041] Grade 2: There are many tumors on the fibrous roots or lateral roots of the root system, while the main root is less affected and slightly swollen.

[0042] Grade 3: The main root of the root system is severely affected and abnormally swollen, and most of the fibrous roots and lateral roots have many tumors.

[0043] Grade 4: The main root of the root system is abnormally swollen, and there are many tumors on the fibrous roots and lateral roots. The total diameter of the root nodules is more than 5 times the diameter of the neck, or the roots are rotten, or even the plant dies.

[0044] S103: Genomic DNA extraction, pooling, and whole-genome sequencing analysis were performed on parents and populations after disease resistance assessment.

[0045] In this step, genomic DNA was extracted from the two groups mentioned above and the parents, a pool was constructed, and whole-genome sequencing was performed. The DNA was then aligned to a reference genome using BWA software, and the ΔSNP-index was determined through SNP calling analysis. Ultimately, RsCr7 was located in a 9.12 Mb region on chromosome R05, thus completing the initial localization of the radish clubroot resistance gene RsCr7.

[0046] Specifically, in the study of mapping the clubroot resistance gene RsCr7 in radish, this application first selected plant samples with two extreme phenotypes from the F2 generation population: 25 resistant (grade 0) and 25 susceptible (grade 4) plants, as well as 20 fresh leaves from each of the two parents. Genomic DNA was extracted from the leaves using a high-efficiency plant genomic DNA extraction kit (DP360, TIANGEN). The DNA from the resistant plants was mixed in equal amounts to form the resistance pool (PR), and the DNA from the susceptible plants was mixed in equal amounts to form the susceptible pool (PS). Subsequently, the genomes of the constructed mixed pools of resistant and susceptible plants, as well as the genomes of the two parents, were sequenced and libraries constructed. Using the radish reference genome RaPsa_xiang-V1.0 (http: / / brassicadb.cn / ) as the reference sequence, clean reads from both parents and the mixed pools of resistant and susceptible radishes were aligned to the reference genome using BWA (v0.7.17) software. SNP calling was performed using SAMtools (v1.9) and Bcftools (v1.9) to obtain SNP loci in each sample. The SNP-index of the resistant and susceptible mixed pools were calculated separately, and the ΔSNP-index between them (i.e., subtracting the SNP-index of the susceptible mixed pool from the resistant pool's SNP-index) was further calculated. Candidate regions for disease resistance genes were determined using sliding window analysis (window size 1Mb, step size 10kb).

[0047] Through significance analysis, this application preliminarily located the radish clubroot resistance gene on chromosome R05 from 1864bp to 9119975bp, covering an area of ​​approximately 9.12Mb (e.g., ...). Figure 2 (As shown). The location interval of this gene is different from the existing location intervals of RsCr1~RsCr3(R09), RsCr4~RsCr5(R08) and RsCr6(R08) (Gan et al.,2019; Gan et al.,2022), which is a new clubroot resistance gene locus. This application names it RsCr7.

[0048] S200: Fine mapping of the clubroot resistance gene RsCr7 in radishes, which was initially mapped, includes the following steps:

[0049] S201: Screening for InDel sites;

[0050] In this step, within the initial localization interval, sites with insertion / deletion (InDel) variations between the two parents are screened out, and sites with insertion / deletion lengths greater than 20 bp are preferentially selected as the objects of subsequent analysis.

[0051] S202: Primer design and PCR validation of the selected InDel sites;

[0052] In this step, primers targeting the selected InDel site were designed using Premier5 software. The effectiveness of these primers was detected by PCR amplification and agarose gel electrophoresis. Primers that showed heterozygosity and significant genotyping effect in the F1 population were screened out (as shown in Table 2) for subsequent F2 population single-plant genotyping.

[0053] Specifically, the PCR amplification system includes: 40 ng DNA, 10 μL 2×Taq PCR MasterMix (KT201, TIANGEN), 0.5 μL 10 μM forward primer, 0.5 μL 10 μM reverse primer, and ddH2O added to a total volume of 20 μL.

[0054] The PCR reaction procedure was as follows: first, pre-denaturation was performed at 94℃ for 4 min; then 32 cycles were performed (denaturation at 94℃ for 30 s, annealing at 57℃ for 30 s, extension at 72℃ for 30 s); finally, extension was performed at 72℃ for 10 min.

[0055] S203: InDel molecular marker typing was performed on individual plants in the F2 population, and genetic linkage analysis was carried out in combination with phenotypic data to draw linkage maps and achieve fine mapping of RsCr7.

[0056] In this step, the selected polymorphic primers were used to perform genotypic analysis on the F2 population, and combined with phenotypic data, genetic linkage analysis and genetic mapping were performed using JoinMap 4.0 software.

[0057] The genotype was recorded as follows: a band matching the resistant parent was recorded as "a", a band matching the susceptible parent as "b", a band matching the F1 band as "h", and a missing band as "-". Based on genotype and phenotypic data from a small population of 500 individuals and a large population of 1506 individuals, RsCr7 was finally located in a region of approximately 259 kb on chromosome R05, specifically between 1046704 bp and 1306128 bp on chromosome R05. The resistance gene was located between the InDel72 and InDel82 markers (e.g., ...). Figure 3 (As shown).

[0058] Table 2 shows the polymorphic InDel primer sequences used for fine-tuning.

[0059]

[0060]

[0061] Based on the above steps, the precise and detailed localization of the radish clubroot resistance gene RsCr7 was achieved.

[0062] S300: Marker development of the finely mapped radish clubroot resistance gene RsCr7, specifically including the following steps:

[0063] S301: Perform bioinformatics analysis on genes within the fine-mapping region and screen RsCr7 candidate genes based on gene annotation information;

[0064] In this step, a total of 56 genes were located within the finely mapped region. Eight genes with non-synonymous mutations or frameshift mutations were selected, and these eight genes were deeply annotated using multiple databases such as KEGG, GO, and COG. The specific eight annotated genes are shown in Table 3.

[0065] Table 3 Gene Information

[0066]

[0067] In Table 3, based on HMMER prediction, the protein encoded by Rsa10043007 has a TIR-NBS-LRR structure, therefore Rsa10043007 is considered the most likely candidate gene for RsCr7.

[0068] S302: PCR amplification, sequencing, and sequence alignment of Rsa10043007 in both resistant and susceptible parent lines revealed 14 InDel and 123 single nucleotide polymorphisms (SNPs) differences between the two lines. Based on one significant 27bp InDel difference, a pair of specific primers (InDel-F and InDel-R) were designed for the development of molecular markers.

[0069] In this step, based on the Rsa10043007 gene sequence in the radish reference genome, forward primers (KL-F: 5'-ATGGATTCTTCTAGTTTATATATG-3') and reverse primers (KL-R: 5'-CTATAAAAATCCCTTATAATATATG-3') for PCR amplification were designed. PCR amplification was then performed using the genomic DNA of the resistant parent 'Snow White' and the susceptible parent 'Sand Pot Radish' based on these forward and reverse primers. The amplification system included: 50 ng DNA, 25 μL SuperHiFi PCRMix (KT212, TIANGEN), 1.5 μL 10 μM forward primer, 1.5 μL 10 μM reverse primer, and ddH2O added to a total volume of 50 μL.

[0070] The PCR reaction procedure includes: first, pre-denaturation at 94℃ for 4 min; then 32 cycles (each cycle includes denaturation at 94℃ for 30 seconds, annealing at 57℃ for 30 seconds, and extension at 72℃ for 3 minutes); and finally extension at 72℃ for 10 minutes to ensure complete extension of all products.

[0071] Furthermore, the amplified PCR products were separated by 1.0% agarose gel electrophoresis, followed by gel excision and recovery of the target fragment. The recovered DNA fragment was ligated into a T-cloning vector, and positive clones were selected and sent to Beijing Qingke Biotechnology Co., Ltd. (Chengdu) for sequencing.

[0072] The full-length sequence of the candidate RsCr7 gene in the resistant parent is 4696 bp (SEQ ID NO.3), while the full-length sequence of the Rscr7 allele in the susceptible parent is 4735 bp (SEQ ID NO.4). The DNAMAN sequence alignment rate is 94.49%. There are 14 InDels and 123 SNPs that differ between the two parents (e.g., Figure 4 (As shown).

[0073] Based on the above information, especially the 27bp sequence difference between the two, this application designed a pair of molecular marker primers (InDel-F: 5'-TATGTTAGTTTCTGCGGCAATAGG-3' (SEQ ID NO.1); InDel-R: 5'-GAAGCTAGGGAAGACTTGGTGT-3' (SEQ ID NO.2)) to distinguish between resistant and susceptible genotypes. PCR amplification experiments were performed on DNA from resistant parents, susceptible parents, F1 generation, and F2 populations. The results showed that the resistant parent produced a 175bp band; the susceptible parent produced a 202bp band; and the F1 generation produced both 175bp and 202bp bands, along with another band of unknown size (e.g., ...). Figure 5 (As shown).

[0074] This application used InDel marker primers established based on the above method to perform PCR amplification on resistant parents, susceptible parents, F1 and F2 populations. The products were detected by agarose gel electrophoresis, and the effectiveness of the marker was evaluated in conjunction with phenotypic data. The comparison showed that the molecular marker achieved 100% consistency with the resistance phenotype of individual plants (as shown in Table 4). This indicates that the molecular marker designed in this application can be effectively used to distinguish individuals carrying different RsCr7 genotypes, providing an efficient and accurate tool for radish clubroot resistance breeding.

[0075] Table 4. Detection of disease-resistant / susceptible samples

[0076]

[0077]

[0078]

[0079] In another exemplary embodiment, to improve the disease resistance of the highly susceptible clubroot cultivar 'Chunbulao', this application employs a marker-assisted selection (MAS) method. Specifically, this involves: using the resistant parent 'Snow White' as the female parent, crossing it with the highly susceptible clubroot cultivar 'Chunbulao' to obtain F1; then, using 'Chunbulao' as the recurrent parent, performing three consecutive backcrosses (BC1 to BC3). In each generation, PCR amplification is performed using the InDel-F / R molecular marker developed based on the RsCr7 gene locus. Materials with heterozygous banding (i.e., simultaneously containing both resistance and susceptibility alleles) are selected as the female parent for the next generation of backcrosses. This process ensures that only plants carrying the target heterozygous resistance gene are selected as the female parent for the next generation. After completing three backcrosses, single plants with the expected heterozygous banding are selected from the BC3F1 population for self-pollination to generate the BC3F2 population. In the BC3F2 population produced by self-pollination, individual plants whose PCR banding patterns were completely identical to those of the resistant parents (i.e., homozygous resistant parental banding patterns) were further screened using PCR amplification products. These individual plants were considered to be 'Chunbulao' materials with successfully improved resistance. Resistance to *Plasmodiophora bacillus* Pb3 was assessed in the obtained improved 'Chunbulao' materials and hybrid combinations prepared from the sterile line *Baiyuanhua* A (e.g., *Baiyuanhua* A / C30519) under indoor conditions. The results showed that the improved 'Chunbulao' and its hybrid combinations all achieved immunity against *Plasmodiophora bacillus* Pb3 (e.g., *Baiyuanhua* A / C30519). Figures 6 to 8 As shown), the results of natural disease identification in the field nursery of the combination white round flower A / C30519 were consistent with the results of indoor identification (as shown). Figure 9 (As shown).

[0080] The above examples not only demonstrate how molecular marker technology can effectively enhance crop disease resistance, but also verify the reliability and practicality of the developed molecular markers. This method allows for the cultivation of new varieties with strong disease resistance in a relatively short time, greatly promoting the sustainable development of agricultural production.

[0081] The above are merely preferred embodiments of this application and do not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. A molecular marker for breeding radish with resistance to clubroot disease, characterized in that, The molecular markers include: Forward primer: 5'-TATGTTAGTTTCTGCGGCAATAGG-3'; Reverse primer: 5'-GAAGCTAGGGAAGACTTGGTGT-3'.

2. A method for constructing molecular markers for clubroot resistance breeding in radishes, characterized in that, The method includes: Preliminary localization of the RsCr7 gene locus for resistance to clubroot disease in radish; Fine mapping was performed on the RsCr7 gene locus, which was initially mapped to resist clubroot disease in radishes. Marker development was carried out on the finely mapped radish clubroot resistance gene locus RsCr7 to obtain molecular markers for radish clubroot resistance breeding.

3. The method for constructing a molecular marker for clubroot resistance breeding of radish according to claim 2, characterized in that, The initial localization of the radish clubroot resistance gene RsCr7 includes: Parental selection and population building; Inoculation experiments and disease resistance assessments were conducted on the selected parents and the constructed populations. Genomic DNA was extracted from parents and populations after disease resistance assessment, pooled sample construction was performed, and whole-genome sequencing analysis was conducted. The genome sequencing data was compared with the reference genome to obtain the SNPs of each sample. The SNP-index of the disease-resistant pool and the disease-susceptible pool and the difference between the two, ΔSNP-index, were calculated to achieve initial localization.

4. The method for constructing a molecular marker for clubroot resistance breeding of radish according to claim 2, characterized in that, The detailed mapping of the clubroot resistance gene RsCr7 in radishes, after initial mapping, includes: Filter InDel sites; Primers were designed and PCR validated for the selected InDel sites; InDel molecular marker typing was performed on individual plants in the F2 population, and genetic linkage analysis was carried out in combination with phenotypic data to draw linkage maps and achieve fine mapping of RsCr7.

5. The method for constructing a molecular marker for clubroot resistance breeding of radish according to claim 2, characterized in that, The marker development of the finely mapped clubroot resistance gene locus RsCr7 in radishes includes: Bioinformatics analysis was performed on genes within the finely mapped regions, and candidate genes for RsCr7 were obtained based on gene annotation information. Based on the candidate gene information, alleles of resistant and susceptible materials were cloned, and molecular markers were developed according to the differences in allele sequences to obtain molecular markers for breeding radish resistance to clubroot disease.

6. The application of a molecular marker for breeding radish with resistance to clubroot disease, characterized in that, The molecular markers are used for breeding disease-resistant varieties.

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