Radish root clubroot resistance kasp marker primer combination, genotype identification method and application

Abstract

This invention relates to the field of plant molecular marker-assisted breeding technology, and discloses a KASP marker primer combination for clubroot resistance in radish, a method for genotyping, and its application. The primer combination includes primers shown in SEQ ID NO:1-3, used to detect the A / G polymorphism at the chr5:48378031 locus in the radish genome, wherein the A allele is linked to clubroot resistance in radish. KASP amplification and collection of FAM and VIC endpoint fluorescence signals are used to determine the AA, A / G, or GG genotype based on clustering results. This method can be used to screen radish breeding materials carrying the A allele, and to screen for A / G genotype materials in backcross generations and AA genotype materials in self-pollinating segregating generations. This invention achieves the detection and screening of resistance-related alleles through specific KASP genotyping, providing a technical means for molecular marker-assisted breeding of radish resistance to clubroot.

Description

Technical Field

[0001] This invention relates to the field of plant molecular marker-assisted breeding technology, specifically to the combination of KASP marker primers for clubroot disease resistance in radish, genotype identification methods, and applications. Background Technology

[0002] Currently, clubroot is a soil-borne disease of cruciferous crops caused by *Platycodon brassicola*, which can infect crops such as radish, cabbage, kale, and rapeseed. After the pathogen invades the root system, it causes swelling of the taproot or lateral roots, affecting the plant's absorption of water and nutrients. Because the pathogen can survive in the soil for a long time, and greenhouse cultivation and continuous cropping increase the likelihood of persistent disease occurrence, clubroot has become one of the most important diseases affecting radish production. Current control methods mainly include chemical control, crop rotation, biological control, and the use of resistant varieties. Among these, the breeding of resistant varieties requires clarifying the genetic basis of resistance and establishing molecular marker detection methods that can be stably used in breeding populations.

[0003] To address the aforementioned need for breeding radish with resistance to clubroot disease, existing research primarily focuses on locating resistance-related regions through genetic population construction, resistance identification, and molecular marker analysis. For example, F2 or F3 strains are constructed using resistant and susceptible materials. 2：3 In population studies, resistance sites are located based on the linkage between resistance and susceptibility phenotypes and molecular markers, and linkage markers such as SSRs are developed for the located regions. Other studies use BSA-seq for sequencing of resistance and susceptibility pools, determining major resistance regions based on allele frequency differences. With the development of SNP genotyping technology, KASP markers can be designed based on single nucleotide polymorphisms flanking or within candidate regions of resistance-related sites. Genotyping is then completed through allele-specific amplification and fluorescence signal detection, and the genotyping results are used for the auxiliary selection of disease-resistant materials.

[0004] While existing clubroot resistance-related loci and molecular markers in radish can provide a basis for resistance genetic analysis, the location intervals of some reported loci remain relatively large, and the genetic basis of resistance differs among different resistance sources, limiting the number of markers available for breeding. Some detection methods are primarily used to determine the presence of resistance-related alleles, and their ability to simultaneously distinguish between homozygous, heterozygous, and susceptible homozygous genotypes still has room for improvement. Phenotypic identification using field disease nurseries or artificial inoculation is also affected by pathogen inoculation, disease development conditions, and the identification cycle. Therefore, it is necessary to establish a molecular detection system targeting specific polymorphic loci related to clubroot resistance in radish that can distinguish between different genotypes, and to use the detection results for the retention of resistance-related alleles in backcross generations and for homozygous screening in self-crossing segregating generations.

[0005] Therefore, this invention provides a combination of KASP marker primers for clubroot disease resistance in radishes, a method for genotyping, and its application, in order to address the shortcomings of existing technologies. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a combination of KASP marker primers for clubroot disease resistance in radishes, a method for genotyping identification, and its application. This solves the problems of the limited number of existing molecular markers for clubroot disease resistance in radishes, the difficulty in accurately distinguishing between homozygous, heterozygous, and homozygous states of resistance-related alleles, and thus limiting the efficiency of marker-assisted screening.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a KASP marker primer combination for clubroot disease resistance in radishes, employing the following technical solution: A KASP marker primer combination for clubroot disease resistance in radishes includes a first allele-specific primer, a second allele-specific primer, and a universal reverse primer.

[0008] The nucleotide sequence of the first allele-specific primer is shown in SEQ ID NO:1, the nucleotide sequence of the second allele-specific primer is shown in SEQ ID NO:2, and the nucleotide sequence of the universal reverse primer is shown in SEQ ID NO:3.

[0009] The primer combination was used to detect the A / G polymorphism at the chr5:48378031 locus in the radish genome. The chr5:48378031 locus was based on the NAU-LB radish reference genome (Project No. PRJCA011486) from the National Genome Science Data Center, where the A allele is an allele linked to clubroot resistance in radish.

[0010] The first allele-specific primer is used to specifically detect the A allele, the second allele-specific primer is used to specifically detect the G allele, and the universal reverse primer is used in conjunction with the first allele-specific primer or the second allele-specific primer to complete the amplification of the target region.

[0011] By employing the above technical solution, two allele-specific primers were designed for the A / G polymorphism at chr5:48378031, and a KASP detection system was established in conjunction with universal reverse primers, enabling different alleles to be identified separately in the same amplification system. This primer combination, based on a defined nucleotide sequence and a defined polymorphic site, can be used for subsequent co-dominance identification of the AA, A / G, and GG genotypes.

[0012] Preferably, the 5′ end of the first allele-specific primer contains a first universal adapter sequence corresponding to the FAM fluorescence channel, and the 5′ end of the second allele-specific primer contains a second universal adapter sequence corresponding to the VIC fluorescence channel; the 3′ ends of the two allele-specific primers respectively contain specific recognition sequences for the A allele and the G allele.

[0013] By adopting the above technical solution, the A allele and the G allele correspond to different fluorescence detection channels, so that the amplification information of the two alleles can be distinguished by different fluorescence signals, providing a detection basis for determining homozygous and heterozygous genotypes.

[0014] Preferably, the first allele-specific primer, the second allele-specific primer, and the universal reverse primer are not directly linked to fluorescent groups; the FAM and VIC fluorescence signals are generated by a fluorescent reporter system in the KASP reaction system that is compatible with the first universal adapter sequence and the second universal adapter sequence.

[0015] By adopting the above technical solution, the universal reverse primer and two allele-specific primers are used together to participate in the amplification of the corresponding alleles, without changing the distinction between different alleles by the FAM fluorescence channel and the VIC fluorescence channel.

[0016] Secondly, this invention provides a KASP method for identifying genotypes related to clubroot disease resistance in radishes, employing the following technical solution: A KASP method for identifying radish genotypes related to clubroot disease resistance, comprising the following steps: The method uses the aforementioned KASP marker primer combination for clubroot disease resistance in radish to identify the genotype of the radish material to be tested. Genomic DNA was extracted from the radish material to be tested.

[0017] The genomic DNA was amplified using the aforementioned KASP marker primer combination for clubroot resistance in radishes.

[0018] The endpoint fluorescence signal of the KASP amplification reaction was collected using the FAM and VIC fluorescence channels. Based on the clustering results of the endpoint fluorescence signal, the A / G polymorphism at the chr5:48378031 site of the radish genome was genotyped to determine whether the radish material to be tested was of AA genotype, A / G genotype, or GG genotype.

[0019] Among them, the FAM fluorescent channel corresponds to the A allele, and the VIC fluorescent channel corresponds to the G allele.

[0020] In the above detection process, the first allele-specific primer corresponds to the A allele at the chr5:48378031 locus, and the second allele-specific primer corresponds to the G allele at the same locus. Through selective amplification using the two allele-specific primers and signal acquisition from the corresponding fluorescence channels, the three genotypes AA, A / G, and GG form different endpoint fluorescence clustering results, thereby achieving codominant genotyping at the chr5:48378031 locus.

[0021] By adopting the above technical solution, the genotype detection of the chr5:48378031 locus can be completed directly using the genomic DNA of the radish material to be tested. It is not necessary to inoculate with clubroot disease and then determine the genotype of this locus based on the disease phenotype. It can also distinguish between homozygous A allele, A / G heterozygous and G allele homozygous status at the same time.

[0022] Preferably, before KASP amplification, the concentration of the genomic DNA is adjusted to 100 ng / μL, and the first allele-specific primer, the second allele-specific primer, and the universal reverse primer are prepared to 10 μM.

[0023] The total reaction volume for KASP amplification was 10 μL. The reaction system included 5 μL of 2×KASP genotyping PCR premix, 1 μL of genomic DNA at a concentration of 100 ng / μL, 0.1 μL of 10 μM first allele-specific primer, 0.1 μL of 10 μM second allele-specific primer, 0.3 μL of 10 μM universal reverse primer, and 3.5 μL of ddH2O.

[0024] By adopting the above technical solution, the amount of the three primers added to the reaction system is matched with the amount of DNA template to be tested, so as to complete the allele-specific amplification and fluorescence typing of the chr5:48378031 locus.

[0025] Preferably, the KASP amplification and fluorescence signal acquisition includes the following steps: Pre-denaturation at 95℃ for 5 minutes; Ten cycles of specific amplification were performed, each cycle consisting of denaturation at 95°C for 15 seconds and annealing for 15 seconds, with the annealing temperature of 61°C in the first cycle and decreasing by 0.6°C in each subsequent cycle. Forty cycles of amplification were performed, each cycle consisting of denaturation at 95°C for 15 seconds and reaction at 55°C for 1 minute, and FAM fluorescence signal and VIC fluorescence signal were collected. The sample was kept at 35°C for 30 seconds, and the FAM fluorescence signal and VIC fluorescence signal were collected at the endpoint.

[0026] By employing the above amplification procedure, the aforementioned gradient annealing process is used for allele-specific amplification, and subsequent cycles are used to amplify the corresponding products and accumulate fluorescence signals to obtain endpoint fluorescence data for genotype clustering analysis.

[0027] Preferably, a two-dimensional scatter plot is generated based on the endpoint FAM fluorescence signal intensity and VIC fluorescence signal intensity, with the FAM fluorescence signal intensity as the Y-axis and the VIC fluorescence signal intensity as the X-axis, and the AA genotype, A / G genotype or GG genotype is determined based on the clustering region of the radish material to be tested in the two-dimensional scatter plot.

[0028] By adopting the above technical solution, the endpoint fluorescence signals corresponding to the two alleles are converted into two-dimensional typing results, which can distinguish the three genotypes AA, A / G and GG in the same detection system, providing a basis for the detection of genotypes related to clubroot disease resistance in radish and the subsequent screening of breeding materials.

[0029] Thirdly, this invention provides an application of a KASP marker primer combination for clubroot resistance in radishes in screening clubroot-resistant breeding materials, employing the following technical solution: Molecular marker-assisted screening of radish breeding materials was performed using the above-mentioned KASP marker primer combination for clubroot resistance in radish, including the following steps: Genomic DNA was extracted from the radish breeding materials to be screened.

[0030] The genomic DNA was amplified using the aforementioned KASP marker primer combination for clubroot resistance in radish, and the A / G polymorphism at the chr5:48378031 site in the radish genome was genotyped.

[0031] Based on the genotyping results, radish materials with the AA or A / G genotype at the chr5:48378031 locus were selected as breeding materials for radish resistance to clubroot carrying the A allele.

[0032] By adopting the above technical solution, the KASP genotyping results at the chr5:48378031 locus can be used for breeding material screening. This allows for the identification of materials carrying the A allele using genomic DNA in the early stages of plant growth, thereby combining the detection of resistance-related alleles with the selection of breeding materials.

[0033] Preferably, the radish breeding material to be screened is a single plant from a backcross generation population. In any backcross generation of backcross breeding, the radish clubroot resistance KASP marker primer combination can be used to genotype the chr5:48378031 locus of the corresponding backcross generation single plant, and A / G genotype single plants can be screened as breeding materials for subsequent backcrosses or self-pollination; in the BC3F1 population formed by the third backcross generation, single plants with the A / G genotype at the chr5:48378031 locus are screened as radish clubroot resistance breeding materials carrying the A allele.

[0034] By adopting the above technical solution, the retention status of the A allele can be detected by KASP genotyping results during continuous backcrossing, and A / G genotype single plants can be selected for subsequent breeding processes, reducing the uncertainty of relying solely on field disease resistance phenotypes for successive generations of screening.

[0035] Preferably, the radish breeding material to be screened also includes the BC3F2 population obtained by self-pollination of single plants with the A / G genotype at the chr5:48378031 locus from the BC3F1 population. The radish clubroot resistance KASP marker primer combination is used to genotype the BC3F2 population at the chr5:48378031 locus, and single plants with the AA genotype are screened as radish clubroot resistance breeding materials homozygous for the A allele. These AA genotype single plants are used to obtain the BC3F3 generation.

[0036] By adopting the above technical solution, the codominant typing of the KASP marker can be used to distinguish between A / G heterozygous genotypes and AA homozygous genotypes in self-crossing segregating populations, and to screen breeding materials in which the A allele has been fixed in homozygosity, thereby realizing the detection, retention and homozygous screening of the A allele in backcross and self-crossing generations.

[0037] In summary, this invention uses the A / G polymorphism at the chr5:48378031 locus in the radish genome as the detection target and establishes a KASP detection system composed of primers shown in SEQ ID NO:1 to SEQ ID NO:3. This system uses the fluorescence channels corresponding to the A and G alleles for endpoint signal acquisition and cluster genotyping, distinguishing between three genotypes: AA, A / G, and GG. Furthermore, the obtained genotype results can be used for marker-assisted screening of radish materials resistant to clubroot disease.

[0038] This invention provides a combination of KASP marker primers for clubroot disease resistance in radishes, a method for genotyping, and its application. It offers the following beneficial effects: 1. This invention designs a KASP marker primer combination targeting the A / G polymorphism at the chr5:48378031 locus in the radish genome. Through the combination of the first allele-specific primer, the second allele-specific primer, and the universal reverse primer, the specific detection of the A allele and the G allele can be achieved. Co-dominance identification of the three genotypes AA, A / G, and GG can be performed, providing a molecular marker basis for the detection of genotypes related to clubroot disease resistance in radish.

[0039] 2. This invention uses KASP amplification and collects the endpoint fluorescence signal through the FAM and VIC fluorescence channels. The genotype at the chr5:48378031 locus is determined based on the clustering results of the fluorescence signals, eliminating the need for clubroot inoculation followed by genotyping based on disease phenotype. In the materials and populations validated by this invention, the K378 genotype showed a good correlation with the clubroot resistance phenotype and can be used for the rapid identification of clubroot resistance-related genotypes in radishes.

[0040] 3. This invention can use the genotyping results of the chr5:48378031 locus to screen breeding materials for radish resistance to clubroot disease. In the backcross generation, A / G genotype materials carrying the A allele are screened, and AA genotype materials are further screened in the self-crossing segregating generation. This enables the detection, retention and homozygous screening of the A allele in the breeding process, providing a technical means for molecular marker-assisted breeding of radish resistance to clubroot disease. Attached Figure Description

[0041] Figure 1 This is a phenotypic diagram of the clubroot resistance of the resistant parent *Acer buergerianum* and the susceptible parent *Andrographis paniculata* after inoculation with *Plasmodiophora brassicae*. Figure 2 This invention presents the process of constructing F1 and F2 populations by hybridizing *Symplocos edulis* and *Chuanxinhong*, along with phenotypic images of some plants. Figure 3 This is a diagram showing the location results of the clubroot resistance association interval of radish obtained based on BSA-QTL analysis in this invention; Figure 4 This is a diagram showing the results of fine-mapping of the clubroot resistance association region of radish using recombinant single plants and SV markers in this invention; Figure 5 This is a fluorescence typing diagram of the KASP molecular marker K378 used in this invention to identify AA, A / G, and GG genotypes at the chr5:48378031 locus. Figure 6 This is a phenotypic diagram of clubroot resistance in individual plants of different K378 genotypes from the BC3F1 backcross population of *Echeveria elegans* in this invention. Figure 7 This is a phenotypic diagram of clubroot resistance in *Radix sclerotiorum* and its improved form in this invention. Figure 8 The phenotypic diagrams of clubroot resistance of Black Leaf Spring and improved Black Leaf Spring in this invention are shown. Figure 9 This is a phenotypic diagram of clubroot resistance in the wine-pot radish and the improved wine-pot radish of the present invention. Detailed Implementation

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] This invention provides a combination of KASP marker primers for clubroot disease resistance in radishes, a method for genotyping, and its application. The following examples are used to illustrate the technical solution and its effects, and are not intended to limit the scope of protection of this invention. Experimental steps not specifically described in the examples were performed using conventional experimental conditions or the conditions for using corresponding reagents and instruments in the art. DNA extraction, PCR product detection, and sequencing data quality control operations not specifically described were all performed using conventional methods in the art.

[0044] The Autumn Snow, Andrographis paniculata, and other radish materials involved in this invention are used for the discovery, localization, or verification of molecular markers. The detection target of the KASP molecular marker K378 in this invention is the genomic DNA of the radish to be tested; the specific detection method does not require Autumn Snow, Andrographis paniculata, or other specific radish materials as a necessary condition.

[0045] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0046] The radish material Qiuxue is a self-pollinated S6 generation material, exhibiting resistance to clubroot disease. It has round, fleshy roots with white skin and flesh, and flat leaves. The radish material Chuanxinhong is an S6 generation red radish material, used as a susceptible parent. Qiuxue and Chuanxinhong were used for disease resistance-related regional mapping and the development of the K378 molecular marker.

[0047] Radish materials used for K378 polymorphism validation included *Caodou* radish, *Sijihong*, *Heiyechunbulao*, *Jiuguan* radish, *Tongzi* radish, *Xijing* radish, *Xinlimei*, *Nanbanzhou*, *Yangzhou Yuanbai*, *Qingcaoba* radish, *Naichai Jiujinwang*, *Huimin Bacunbai*, *Xiaoshan Yidaozhong*, *Biangan Hong*, *Lubafen*, and *Zhongtui* radish. All of these materials were from generation S6, and their clubroot resistance and morphological characteristics are shown in Table 5. These materials were used to validate the K378 polymorphism and marker suitability.

[0048] Brassica clubroot fungus ( Plasmodiophora brassicae Woron. The inoculum was used for clubroot disease resistance identification in radishes. When constructing the F2 population, the effective spore concentration of the inoculum used for supplementary inoculation was 10-1. 7 per g.

[0049] K378-FAM, K378-VIC, and K378-R are all oligonucleotide primers. K378-FAM is a first allele-specific primer, K378-VIC is a second allele-specific primer, and K378-R is a universal reverse primer. The nucleotide sequence of K378-FAM is shown in SEQ ID NO:1, with a universal adapter sequence corresponding to the FAM fluorescent channel at its 5′ end; the nucleotide sequence of K378-VIC is shown in SEQ ID NO:2, with a universal adapter sequence corresponding to the VIC fluorescent channel at its 5′ end; and the nucleotide sequence of K378-R is shown in SEQ ID NO:3. None of these three primers directly connect to fluorescent groups; K378-FAM and K378-VIC are primer names used to easily distinguish the corresponding fluorescent channels. When used in PCR reactions, the stock solution concentration for all three primers is 10 μM. After adding 10 μL of the reaction system according to the present invention, the final concentrations of K378-FAM, K378-VIC and K378-R in the reaction system are 0.1 μM, 0.1 μM and 0.3 μM, respectively.

[0050] 2×KASP PCR Mix is ​​a commercially available PCR premix for KASP genotyping, specifically used for allele-specific amplification and fluorescence typing of the K378 molecular marker; 2×PCR Mix is ​​a commercially available conventional PCR premix for SV marker amplification. The specific commercial formulations of the above premixes do not constitute a limitation of the technical solution of this invention.

[0051] Agarose, molecular biology grade, CAS number 9012-36-6, is used for gel electrophoretic separation of SV-labeled PCR amplification products, with a concentration of 0.8%–1.5%. Agarose is a commonly available commercially available molecular biology experimental material.

[0052] Preparation Example 1: This preparation example provides a method for preparing resistant and susceptible mixed pools for analysis of clubroot disease resistance-related regions in radishes, including the following steps: Autumn Snow, which exhibits stable resistance to clubroot, was selected as the resistant parent, and Andrographis paniculata was selected as the susceptible parent. Clubroot inoculation tests were performed on both parents, and the results are as follows: Figure 1 As shown, no clubroot phenotype was observed after inoculation with *Euphorbia milii*, while the fleshy root enlargement was inhibited after inoculation with *Cutrocus sativus*, and pathological masses formed on the main root and lateral roots.

[0053] Crossing was performed using *Sedum aubergine* as the female parent and *Corydalis yanhusuo* as the male parent to obtain F1 generation seeds; F1 generation plants were then self-pollinated to obtain F2 generation seeds. The population construction process and some F1 and F2 plant phenotypes are as follows: Figure 2 As shown.

[0054] Eighty hundred F2 generation seeds were selected and sown in a diseased nursery in an area affected by clubroot disease, and supplemented with an effective spore concentration of 10. 7 Clubroot inoculum was prepared at a concentration of 1 / g. The incidence of clubroot disease in individual plants was assessed after 60 days of plant growth.

[0055] Fifty disease-free individuals were selected from the F2 population to form a resistant mixed pool (R-pool), and extremely susceptible individuals were selected to form a susceptible mixed pool (S-pool). Genomic DNA was extracted from *Symplocos edulis*, *Andrographis paniculata*, R-pool, and S-pool for subsequent sequencing and BSA-QTL analysis.

[0056] Example 1: This embodiment provides primer combinations, genotype identification methods, and applications for KASP markers for clubroot resistance in radishes. Specifically, it involves the development process of the KASP molecular marker K378. Through disease resistance genetic region localization, fine mapping of recombinant single plants, candidate region sequence differential analysis, and disease resistance-related SNP screening, the chr5:48378031 locus was determined, and the KASP molecular marker K378 was developed for this locus, including the following steps: Genomic DNA was extracted from Qiuxue, Chuanxinhong, R-pool, and S-pool in Preparation Example 1 and sequenced. The raw sequencing data, after quality control and filtering, was compared with the radish reference genome NAU-LB, whose project number in the National Genome Science Data Center is PRJCA011486. ​​The chromosome coordinates of the chr5:48378031 locus in this specification are based on the NAU-LB radish reference genome of the National Genome Science Data Center project number PRJCA011486. ​​Polymorphic SNP loci between the two parents were screened, and BSA-QTL analysis was performed using the Δ(SNP-index) algorithm. A sliding window was used to smoothly fit the Δ(SNP-index) algorithm, with a window size of 1000kb and a step size of 10kb. The localization results are as follows: Figure 3 As shown, in the *Radix augrass* × *Radix Andrographis paniculata* F2 population, a region significantly associated with clubroot resistance was obtained on chromosome 5 of radish, and this major-effect QTL resistance locus was denoted as... RsCr8 At a 99% confidence level, the correlation interval is located at chr5:43250001-49559736bp, with a physical interval of approximately 6.3Mb.

[0057] Within the chr5:43.25–49.56 Mb association interval, we analyzed the insertion or deletion sequence differences of 100–500 bp between the genomes of *Symplocos edulis* and *Hemiberlesia lataniae*. Specific primers were designed at both ends of the differentially expressed regions to amplify PCR fragments of 300–800 bp in length, and the SV markers shown in Table 1 were obtained.

[0058] Table 1. SV markers and primers used for fine mapping ; Each SV marker was amplified using the PCR reaction system shown in Table 2. The amplification products were detected by 0.8%–1.5% agarose gel electrophoresis. Amplification bands that matched the resistant parent *Symplocos edulis* were denoted as A, those that matched the susceptible parent *Andrographis paniculata* were denoted as B, those that had bands from both parents were denoted as H, and those that did not obtain amplification bands were denoted as "-".

[0059] Table 2. SV-labeled PCR amplification reaction system ; The PCR reaction procedure is shown in Table 3.

[0060] Table 3. SV-labeled PCR amplification reaction procedure ; The linkage between clubroot resistance phenotypes and SV marker genotypes in 708 F2 single plants was analyzed. Based on 8 F2 single plants that underwent recombination exchange, the target region associated with clubroot resistance was narrowed down to chr5:48.267–48.556 Mb between GZ032 and GZ012, with a region length of approximately 289 kb.

[0061] Further analysis of 1156 F2 plants yielded four recombinant exchange plants through screening with GZ032 and GZ012. Sequence differences in these recombinant exchange plants within the range of 48.267–48.556 Mb were analyzed, further narrowing the target region to chr5: 48.378–48.457 Mb, with a region length of approximately 79 kb. The fine mapping process and genotypes of the recombinant plants are detailed below. Figure 4 . Figure 4 The 79kb interval is defined by the boundaries of 48.378Mb and 48.457Mb.

[0062] Genes within the 79kb candidate region were analyzed based on the NAU-LB reference genome annotation. The reference genome annotation yielded 16 genes from Rsa5g054370 to Rsa5g054520. Homology alignment revealed one additional unannotated gene, Novel1, located between Rsa5g054420 and Rsa5g054430. The annotation results for each gene are shown in Table 4.

[0063] Table 4. Gene analysis results within candidate intervals ; ; Note: "—" indicates that no corresponding remarks were recorded.

[0064] Rsa5g054450 and Rsa5g054460 were both absent in both the resistant and susceptible parents; Rsa5g054370, Rsa5g054380, Rsa5g054420, Rsa5g054470, Rsa5g054490, Rsa5g054510, and Rsa5g054520 were not expressed in the roots of either parent. Based on the gene functional annotations within the candidate regions and the sequence differences between the two parents, [the following was considered]: Novel1 Novel1 is a candidate gene for resistance to clubroot disease. It is completely absent in susceptible parents but present in resistant parents, and its function is predicted to be a TIR-NBS-LRR family-related protein.

[0065] Because the susceptible parents have candidate genes Novel1 The region contains a complete deletion. To obtain a codominant molecular marker that can distinguish between homozygous resistant, heterozygous resistant, and homozygous susceptible genotypes, [the following was studied]. Novel1 The 10kb regions immediately upstream and downstream of the 5′ and 3′ ends were used as SNP screening regions to compare sequencing data from different radish germplasm resources. Table 5 lists radish germplasm materials from different sources, showing differences in root shape, skin color, and leaf type. SNPs in the target region of these materials were genotyped, and the chr5:48378031 locus was obtained. This locus is genotype AA in the resistant parent Qiuxue, and genotype GG in the susceptible materials tested (see Table 5 for details). Combined with subsequent verification results from F2 populations, backcross populations, and different genetic backgrounds, the A allele at the chr5:48378031 locus is an allele linked to radish clubroot resistance.

[0066] Table 5. SNP genotyping results at the chr5:48378031 locus in different radish materials. ; ; A KASP molecular marker, K378, was designed targeting the A / G polymorphism site at chr5:48378031. K378 comprises allele-specific primers K378-FAM and K378-VIC, and a universal reverse primer K378-R. The nucleotide sequences of K378-FAM, K378-VIC, and K378-R are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. In this invention, K378-FAM is also referred to as the first allele-specific primer, K378-VIC as the second allele-specific primer, and K378-R as the universal reverse primer. The first allele-specific primer is used to specifically detect the A allele, and the second allele-specific primer is used to specifically detect the G allele.

[0067] The 5′ end of K378-FAM is a universal adapter sequence corresponding to the FAM fluorescence channel, and its 3′ allele-specific recognition region is used to recognize the A allele at chr5:48378031. The 5′ end of K378-VIC is a universal adapter sequence corresponding to the VIC fluorescence channel, and its 3′ allele-specific recognition region is used to recognize the G allele at chr5:48378031. K378-R serves as a reverse primer shared by K378-FAM and K378-VIC. None of the three primers are directly linked to fluorescent groups during synthesis. During KASP amplification, the 5′ universal adapter sequence, together with the accompanying fluorescent reporter system in the reaction system, enables the detection of the corresponding FAM or VIC fluorescence signal.

[0068] When the chr5:48378031 site of the DNA sample contains the A allele, the primer shown in SEQ ID NO:1 undergoes allele-specific amplification and generates a FAM fluorescent signal through a fluorescent reporter system compatible with its 5′ universal adapter sequence. When the site contains the G allele, the primer shown in SEQ ID NO:2 undergoes allele-specific amplification and generates a VIC fluorescent signal through a fluorescent reporter system compatible with its 5′ universal adapter sequence. The DNA sample containing only the A allele is classified as AA genotype; the DNA sample containing both the A and G alleles is classified as A / G genotype; and the DNA sample containing only the G allele is classified as GG genotype.

[0069] The above-mentioned disease resistance genetic region localization, recombination and exchange analysis, and the SV markers shown in Table 1 were used to determine the screening range of the K378 target site and to verify its localization. The final detection target for KASP genotyping was the A / G polymorphic site at chr5:48378031 in the NAU-LB reference genome, detected using the three primers shown in SEQ ID NO:1 to SEQ ID NO:3. RsCr8 The associated interval and the SV marker primers shown in SEQ ID NO:4 to SEQ ID NO:23 are used for the development and localization verification of K378, but are not a necessary component for genotyping using K378.

[0070] Example 2: This embodiment provides a combination of KASP marker primers for clubroot resistance in radish, a method for genotyping, and its application. Specifically, it involves a method for codominant genotyping of the A / G polymorphic site at chr5:48378031 in radish using K378. The method involves detecting the radish genomic DNA using three primers shown in SEQ ID NO:1 to SEQ ID NO:3, and determining the AA, A / G, and GG genotypes based on FAM and VIC fluorescence signals. The steps include: Genomic DNA was extracted from the radish material to be tested, using a DNA template concentration of 100 ng / μL. Before KASP amplification, the concentration of genomic DNA was adjusted to 100 ng / μL, and the first allele-specific primer K378-FAM, the second allele-specific primer K378-VIC, and the universal reverse primer K378-R were prepared to a concentration of 10 μM. KASP amplification was performed using K378-FAM (SEQ ID NO:1), K378-VIC (SEQ ID NO:2), and K378-R (SEQ ID NO:3), with a total reaction volume of 10 μL. The specific reaction system is shown in Table 6.

[0071] Table 6. K378 molecular marker KASP amplification reaction system ; Perform PCR amplification and fluorescence signal acquisition according to the procedure shown in Table 7.

[0072] Table 7. KASP amplification and fluorescence acquisition procedure for K378 molecular marker ; Specifically, KASP amplification and fluorescence signal acquisition include: pre-denaturation at 95℃ for 5 min; 10 cycles of specific amplification, each cycle including denaturation at 95℃ for 15 s and annealing for 15 s, with the annealing temperature of 61℃ in the first cycle and decreasing by 0.6℃ in each subsequent cycle; 40 cycles of amplification, each cycle including denaturation at 95℃ for 15 s and reaction at 55℃ for 1 min, and acquisition of FAM and VIC fluorescence signals; and holding at 35℃ for 30 s and acquisition of the endpoint FAM and VIC fluorescence signals.

[0073] PCR amplification and FAM and VIC fluorescence signal acquisition were performed using a real-time fluorescence PCR instrument, and genotyping was performed using the accompanying genotyping software. Fluorescence signals from the KASP amplification reaction were acquired through the FAM and VIC fluorescence channels, where the FAM channel corresponds to the A allele and the VIC channel corresponds to the G allele. A two-dimensional scatter plot was generated based on the fluorescence signal intensity at the PCR endpoint, with the FAM fluorescence signal intensity as the Y-axis and the VIC fluorescence signal intensity as the X-axis.

[0074] Genotypes of the samples were determined based on two-dimensional scatter plots and clustering results of endpoint fluorescence signals. Genotyping was performed on the A / G polymorphism at the chr5:48378031 locus in the radish genome. Samples with predominant FAM signal were identified as AA genotypes; samples with predominant VIC signal were identified as GG genotypes; and samples with strong FAM and VIC signals were identified as A / G heterozygous genotypes. Typical genotyping results for K378 are shown below. Figure 5 As shown.

[0075] The clubroot resistance of radish materials was predicted based on the K378 genotype. In the materials and populations verified in this invention, the AA genotype was determined to carry a homozygous resistance allele, the A / G genotype was determined to carry a heterozygous resistance allele, and the GG genotype was determined to be a homozygous susceptible genotype.

[0076] Example 3: This embodiment provides a KASP marker primer combination, genotype identification method, and application for clubroot resistance in radishes. Specifically, it involves the application of the KASP molecular marker K378 in backcross-assisted selection for clubroot resistance in four-season red radishes, including marker-assisted screening of the A resistance allele at chr5:48378031 in the backcross generation, and verification of K378 predicted resistance and field clubroot resistance, including the following steps: In this embodiment, the radish breeding material to be screened is a single plant from the backcross generation population.

[0077] The F1 generation was obtained by crossing the disease-resistant parent with the disease-susceptible Four Seasons Red Radish, and then backcrossing was performed using the Four Seasons Red Radish as the recurrent parent.

[0078] Genomic DNA was extracted from the seedlings of each backcross generation. Genotyping was performed using K378 according to the method in Example 2. Individual plants with the K378 genotype A / G were selected for further backcrossing with Four Seasons Red Radish. That is, in three consecutive backcross generations, K378 was used to genotype the chr5:48378031 locus of individual plants in each backcross generation, and individual plants with the A / G genotype were selected from each backcross generation as breeding materials for the next backcross generation.

[0079] The BC3F1 population was obtained through three consecutive backcrosses. From the BC3F1 population formed in the third backcross generation, 46 individual plants were selected for K378 genotyping and field identification for clubroot disease. Among these, 22 plants with the K378 genotype A / G were used as marker-assisted selection plants carrying the A resistance allele; specifically, plants with the A / G genotype at the chr5:48378031 locus were selected as breeding materials for radish resistance to clubroot carrying the A allele.

[0080] The above-mentioned BC3F1 individual plants were sown in a field disease nursery for clubroot resistance assessment. The measured resistance of each individual plant was recorded, and the predicted resistance of K378 was compared with the measured resistance in the field. Root phenotypes of some individual plants are shown below. Figure 6 .

[0081] Example 4: This embodiment provides a KASP marker primer combination, genotype identification method, and application for clubroot resistance in radishes. Specifically, it involves the application of the KASP molecular marker K378 in improving clubroot resistance in different radish genetic backgrounds, including backcrossing of grass-root radish, black-leaf spring radish, and wine-pot radish, K378-assisted selection, and homozygous fixation of the A resistance allele at chr5:48378031. The steps include: Using grass-root radish, black-leaf spring radish, and wine-jar radish as susceptible recurrent parents, respectively, they were crossed with disease-resistant parents to obtain the corresponding F1 generation.

[0082] Three consecutive generations of backcrossing were performed using *Radish simonii*, *Radish 'Black Leaf Spring'*, and *Radish 'Wine Pot'* as recurrent parents. For each backcross generation, the genotype of the offspring was detected using the K378 assay as described in Example 2. Individual plants with A / G genotypes were selected for further backcrossing to obtain the corresponding BC3F1 population.

[0083] In this embodiment, the radish breeding materials to be screened also include the BC3F2 population obtained by self-pollination of single plants with the A / G genotype at chr5:48378031 locus from the BC3F1 population. Single plants with the A / G genotype were selected for self-pollination in the BC3F1 generation to obtain the BC3F2 population. Homozygous AA plants were then screened using K378, and these homozygous AA plants were further self-pollinated to obtain the BC3F3 generation, yielding improved grass-root radish, improved black-leaf spring-growing radish, and improved wine-pot radish, respectively. AA genotype single plants were used as breeding materials for radish resistance to clubroot disease with homozygous A alleles, and were used to obtain the BC3F3 generation.

[0084] Genotypes of unmodified *Radish simonii*, *Radish 'Black Leaf Spring', and *Radish 'Wine Pot'*, as well as the corresponding modified material K378 genotype, were tested, and clubroot resistance was identified. Root phenotypes of *Radish simonii* and the modified *Radish simonii* are shown below. Figure 7 The root phenotypes of *Heterophyllum burgundy* and improved *Heterophyllum burgundy* are shown in [reference needed]. Figure 8 The root phenotype of the wine-potted radish and the improved wine-potted radish is shown in [reference needed]. Figure 9 .

[0085] Comparative Example 1: Compared with Example 3, the difference is that 24 individual plants with the K378 genotype GG from the same BC3F1 backcross population were selected as controls. These 24 individual plants did not carry the A resistance allele at the chr5:48378031 locus. The other field nursery resistance identification methods were the same.

[0086] Comparative Example 2: Compared with Example 4, the difference is that the test materials are grass radish, black leaf spring old man radish and wine radish that have not been backcrossed for disease resistance improvement. Ten plants of each material were tested, and their K378 genotype was GG. The other methods for clubroot resistance identification were the same.

[0087] Test Example 1: This test case is used to verify the fine localization of the candidate region. Novel1 The feasibility of screening for disease resistance-related SNPs at chr5:48378031 based on structural differences and establishing a K378 codominant genotyping system based on this locus was investigated, and the correspondence between the K378 genotype and the clubroot resistance phenotype in the F2 population was verified.

[0088] BSA-QTL analysis was performed on the F2 population obtained in Preparation Example 1 according to the method in Example 1. Figure 3 The Δ (SNP-index) results shown record the association interval of clubroot resistance on chromosome 5 of radish.

[0089] Recombination and recombination analysis was performed on the F2 population using the SV markers shown in Table 1. First, the genotype and phenotypic relationships of 8 recombination monofilaments out of 708 F2 monofilaments were recorded, and an approximately 289 kb interval between GZ032 and GZ012 was determined based on the recombination breakpoint. Then, 1156 F2 monofilaments were screened, and 4 monofilaments for further recombination and recombination were recorded. Based on the recombination breakpoint, the candidate interval was limited to chr5: 48.378–48.457 Mb. For fine mapping results, please refer to [reference needed]. Figure 4 .

[0090] Genes within the candidate regions were analyzed according to the method in Example 1. Candidate genes. Novel1 It exists in resistant parents but is absent in susceptible parents. Based on this structural difference, [the following is discussed]: Novel1 SNP screening was performed on the 10kb upstream and downstream regions immediately adjacent to the 5′ and 3′ ends of the radish, and the chr5:48378031 locus of the radish materials listed in Table 5 was genotyped to record the allele status of the resistant radish and each susceptible material.

[0091] Following the method in Example 2, KASP typing was performed on 94 F2 individual plants using K378, and the field clubroot resistance phenotype of each individual plant was recorded. Resistance was statistically analyzed according to the AA, A / G, and GG genotypes. The KASP fluorescence typing results are available in [reference needed]. Figure 5 The specific test data are shown in Table 8.

[0092] Table 8. Genotypes of K378 and results of clubroot resistance verification in 94 F2 individual plants. ; ; Note: "—" indicates that there is no corresponding F2 single plant and corresponding test data at this position.

[0093] in conclusion: According to the data in Table 8, among the 94 F2 individual plants, 21 plants were of the AA genotype, all of which showed disease resistance; 55 plants were of the A / G genotype, all of which showed disease resistance; and 18 plants were of the GG genotype, all of which showed susceptibility. The K378 genotype of all 94 individual plants matched the experimental phenotype of clubroot, with a genotype-phenotype consistency rate of 100%.

[0094] according to Figure 3 and Figure 4 The disease resistance-associated region was gradually narrowed down through BSA-QTL analysis and recombination exchange analysis, from an initial association range of approximately 6.3 Mb to a 289 kb interval between GZ032 and GZ012, and further narrowed to a 79 kb interval of chr5: 48.378–48.457 Mb. This localization process was used to limit the scope of subsequent candidate gene analysis and SNP screening.

[0095] According to Table 4 Novel1 It exists in resistant parents but is absent in susceptible parents. Based on this structural difference, in Novel1 SNPs suitable for co-dominance detection were further screened in neighboring areas. According to Table 5, the chr5:48378031 locus was AA in the resistant material Qiuxue, while all susceptible materials listed in Table 5 were GG. The tested materials showed differences in root shape, skin color, and leaf type. In the radish materials covered in this test, the chr5:48378031 locus genotype consistently corresponded to the clubroot resistance phenotype.

[0096] according to Figure 5Using primers K378 (SEQ ID NO:1–SEQ ID NO:3) to detect the A / G polymorphism at chr5:48378031, three distinguishable genotyping regions were formed by FAM and VIC fluorescence signals. The region dominated by FAM signal corresponds to the AA genotype, the region with strong FAM and VIC signals corresponds to the A / G genotype, and the region dominated by VIC signal corresponds to the GG genotype. Combined with the data in Table 8, AA and A / G plants all showed resistance, while GG plants all showed susceptibility. K378 can distinguish between homozygous resistant, heterozygous resistant, and homozygous susceptible genotypes in the same detection system.

[0097] Fine-tuning of candidate intervals Novel1 Structural difference analysis, multi-material screening at the chr5:48378031 locus, and genotypic and phenotypic verification results of 94 F2 single plants show that the K378 co-dominant detection system established for the A / G polymorphism at chr5:48378031 can be used for the detection of clubroot resistance-related genotypes in the radish materials of this invention.

[0098] Test Example 2: In this test case, the A / G genotype BC3F1 plant carrying the A disease resistance allele in Example 3 was used as the experimental group, and the GG genotype plant in the same BC3F1 population in Comparative Example 1 was used as the control group. This was used to verify the auxiliary selection effect of K378 on disease-resistant plants in the backcross population and the consistency between the marker-predicted resistance and the resistance measured in the disease nursery.

[0099] K378 genotyping was performed on 46 BC3F1 individual plants according to the method in Example 2.

[0100] Based on the K378 genotype, the 46 individual plants were divided into the A / G genotype group and the GG genotype group, with 22 plants of the A / G genotype and 24 plants of the GG genotype. The A / G genotype was predicted to be resistant, while the GG genotype was predicted to be susceptible.

[0101] Forty-six BC3F1 plants were placed in a clubroot disease nursery for resistance identification, and the measured resistance of each plant was recorded.

[0102] The predicted resistance of K378 was compared with the actual resistance measured in the disease nursery on a plant-by-plant basis. The results are shown in Table 9. For representative root phenotypes, please refer to [reference needed]. Figure 6 .

[0103] Table 9. Genotypes, predicted resistance, and measured resistance of the K378 genotype in the BC3F1 backcross population. ; ; in conclusion: According to the data in Table 9, among the 46 BC3F1 plants, all 22 plants with the A / G genotype showed resistance, while all 24 plants with the GG genotype showed susceptibility. The predicted results of the K378 marker for all 46 plants were consistent with the actual results measured in the disease nursery, with a consistency rate of 100%.

[0104] Example 3 and Comparative Example 1 were derived from the same BC3F1 backcross population. The difference between them in the K378 detection results was the different alleles carried at the chr5:48378031 locus. A / G genotype plants carried the A resistance allele and exhibited resistance in the disease nursery; GG genotype plants did not carry the A resistance allele and were susceptible in the disease nursery. The detection results within the same backcross population indicate that K378 can be used to screen for plants carrying the A resistance allele.

[0105] according to Figure 6 In the BC3F1 population, the fleshy roots of A / G genotype plants remained normally developed, while GG genotype plants exhibited clubroot disease phenotype. Figure 6 The root phenotypes shown are consistent with the results of individual plant testing in Table 9.

[0106] According to Table 9 and Figure 6 In the BC3F1 population used in this test, K378's identification of the A / G and GG genotypes corresponded to the respective disease-resistant and susceptible phenotypes. As a co-dominant marker, K378 could also distinguish the homozygous AA genotype. Using K378 to screen for A / G genotype plants during backcrossing preserved the A disease-resistant allele, and subsequent screening for AA genotype plants in self-pollinating segregating generations achieved homozygous fixation of the A disease-resistant allele.

[0107] Test Example 3: This test case uses the improved grass-root radish, improved black-leaf spring radish, and improved wine-jar radish obtained in Example 4 as experimental materials, and the unmodified grass-root radish, black-leaf spring radish, and wine-jar radish in Comparative Example 2 as control materials, to verify the genotyping effect of K378 in different radish genetic backgrounds and its applicability in molecular marker-assisted breeding for clubroot resistance.

[0108] Ten plants each of the following varieties were selected: Autumn Snow, Grassroot Radish, Improved Grassroot Radish, Black Leaf Spring Old, Improved Black Leaf Spring Old, Wine Pot Radish, and Improved Wine Pot Radish, and genomic DNA was extracted.

[0109] The K378 genotype of each individual plant was detected according to the method in Example 2. Clubroot resistance was predicted based on the K378 genotype, with the AA genotype predicted as resistant and the GG genotype as susceptible.

[0110] Clubroot resistance was assessed for each material, and the measured resistance was recorded. See also: *Radish stalk* and improved *Radish stalk*. Figure 7For reference on Black Leaf Spring Everlasting and Improved Black Leaf Spring Everlasting, please see [link / reference]. Figure 8 See also: Sake Jar Radish and Improved Sake Jar Radish Figure 9 .

[0111] The predicted resistance and actual resistance of K378 were compared plant by plant, and the results are shown in Table 10.

[0112] Table 10. Results of K378 genotype and clubroot resistance verification in different radish materials ; ; in conclusion: According to the data in Table 10, all 10 resistant parent plants, *Qiusue*, were of the AA genotype and exhibited resistance. The remaining 30 plants (10 each of the unmodified *Caodou*, *Heiye Chunbulao*, and *Jiuguan* varieties) were of the GG genotype and exhibited susceptibility. The corresponding 30 modified *Caodou*, *Heiye Chunbulao*, and *Jiuguan* varieties were all of the AA genotype and exhibited resistance. The predicted resistance of K378 in all 70 plants matched the measured resistance.

[0113] according to Figure 7 Unmodified radish roots showed root infection and abnormal development, while modified radish roots maintained normal development; according to Figure 8 Unmodified *Heiyechun Bulao* exhibited clubroot disease phenotypes, while modified *Heiyechun Bulao* showed normal fleshy root development; according to Figure 9 Unmodified wine-potted radishes showed obvious pathological swelling and abnormal development of the roots, while modified wine-potted radishes did not show the corresponding disease phenotype.

[0114] Example 4 and Comparative Example 2 used three different radish genetic backgrounds—grassroots radish, black-leaf spring radish, and wine-jar radish—for verification. All three unmodified materials were GG genotype and showed susceptibility to disease. After continuous backcrossing and using K378 to retain A / G single plants, followed by self-pollination to select AA homozygous single plants, all three improved materials obtained the AA genotype and showed disease resistance. According to Table 10 and... Figures 7-9 In the different genetic backgrounds verified by this invention, the chr5:48378031 genotype obtained by K378 detection has a consistent correspondence with the actual phenotype of clubroot disease, and can be used for the detection and fixation of A resistance alleles in backcross and selfcross generations.

[0115] Combining the results of test examples 1 to 3, in the candidate genes Novel1 In a genetic context present in resistant parents but absent in susceptible parents, through the analysis of... Novel1Multi-material SNP screening was conducted in the vicinity to identify the A / G polymorphic site at chr5:48378031, and a K378 codominant KASP genotyping system was established based on this. K378 was distinguished into three genotypes—AA, A / G, and GG—using specific primers shown in SEQ ID NO:1–SEQ ID NO:3, and FAM and VIC fluorescence signals. In 94 F2 plants, the K378 genotype showed 100% concordance with the experimental clubroot phenotype. In 46 backcrosses of the Sijihong BC3F1 genotype, 22 A / G plants showed resistance, and 24 GG plants showed susceptibility. In three genetic backgrounds—grassroots radish, black-leaf spring radish, and wine-pot radish—unmodified GG genotype materials showed susceptibility, while AA genotype materials obtained after backcrossing and homozygous fixation of the A allele showed resistance.

[0116] The above results indicate that the A / G polymorphism at chr5:48378031, upon which K378 detection is based, can be used to distinguish genotypes associated with clubroot resistance in the F2 segregating population, BC3F1 backcross population, and different radish genetic backgrounds verified in this invention. Using K378, plants carrying the A allele can be screened through genomic DNA detection at the seedling stage. During backcrossing, A / G genotype plants are selected for continuous transgenic A resistance allele. In the self-pollinating segregating generation, AA genotype plants are selected to complete homozygous fixation of the A resistance allele, thus enabling marker-assisted selection for clubroot resistance in radishes. Specifically, the KASP marker primer combination for clubroot resistance in radishes can be used for marker-assisted screening of radish breeding materials. Based on the genotyping results at the chr5:48378031 locus, AA genotype or A / G genotype radish materials can be screened as clubroot resistance breeding materials carrying the A allele.

[0117] Appendix: SEQ ID NO:1 (K378-FAM primer): 5′-GAAGGTGACCAAGTTCATGCTAGCTTTTTAATACACACAGAGAAGT-3′ SEQ ID NO:2 (K378-VIC primer): 5′-GAAGGTCGGAGTCAACGGATTAGCTTTTTTAATACACACAGAGAAGC-3′ SEQ ID NO:3 (K378-R primer): 5′-GTCATTTCGGCGTCAATCTTCTTCC-3′ SEQ ID NO:4 (GZ005 forward primer): 5′-CACATGAATAGATGCTAGTGGTG-3′ SEQ ID NO:5 (GZ005 reverse primer): 5′-GAAGGCTACAACGGGTAAAAG-3′ SEQ ID NO:6 (GZ008 forward primer): 5′-GAGAGCATTGGTATATCTGAAGAAG-3′ SEQ ID NO:7 (GZ008 reverse primer): 5′-GAACCAATAGTCTGTGTACACATTC-3′ SEQ ID NO:8 (GZ032 forward primer): 5′-GAGAGAGACGTAGTAGATGCTAGG-3′ SEQ ID NO:9 (GZ032 reverse primer): 5′-CAACTGGCCTCTGTATCTTATTTG-3′ SEQ ID NO:10 (GZ012 forward primer): 5′-CCGTTCATTCTTCTTCGTCGTC-3′ SEQ ID NO:11 (GZ012 reverse primer): 5′-CTTCTGGTGTCTAATTTGTCTGATG-3′ SEQ ID NO:12 (GZ017 forward primer): 5′-CCACTGTTATTGAACTTGAATTTC-3′ SEQ ID NO:13 (GZ017 reverse primer): 5′-GTTTCTGGTTGTTTTGTTTTCTGC-3′ SEQ ID NO:14 (GZ019 forward primer): 5′-GTTGGTGAGTCACTTGGTATGAG-3′ SEQ ID NO:15 (GZ019 reverse primer): 5′-CTGTCCACTCGAGAAATTAGTTTG-3′ SEQ ID NO:16 (GZ018 forward primer): 5′-GCTCCAAGCTATCAAAACTTACC-3′ SEQ ID NO:17 (GZ018 reverse primer): 5′-GAACTAGAAAAGTAATCTCAAAGGTAC-3′ SEQ ID NO:18 (GZ022 forward primer): 5′-GACTCGGACAAGACTCACCATG-3′ SEQ ID NO:19 (GZ022 reverse primer): 5′-GGGCTTGATGTTTTTACATGATTC-3′ SEQ ID NO:20 (GZ028 forward primer): 5′-GGCAACTGTGAATCTATGAATCC-3′ SEQ ID NO:21 (GZ028 reverse primer): 5′-CCCGGCATTAAGCGTTTTTCTC-3′ SEQ ID NO:22 (GZ025 forward primer): 5′-CCTTACTCATCTGCAGGTGCTTGG-3′ SEQ ID NO:23 (GZ025 reverse primer): 5′-CGTTGTTCTCGATTCTTTCCTC-3′

Claims

1. A KASP-labeled primer combination for clubroot disease resistance in radishes, characterized in that, This includes first allele-specific primers, second allele-specific primers, and universal reverse primers; The nucleotide sequence of the first allele-specific primer is shown in SEQ ID NO:1, the nucleotide sequence of the second allele-specific primer is shown in SEQ ID NO:2, and the nucleotide sequence of the universal reverse primer is shown in SEQ ID NO:

3. The primer combination was used to detect the A / G polymorphism at the chr5:48378031 locus in the radish genome. The chr5:48378031 locus was based on the NAU-LB radish reference genome with the National Genome Science Data Center project number PRJCA011486, where the A allele is an allele linked to resistance to clubroot disease in radish. The first allele-specific primer is used to specifically detect the A allele, and the second allele-specific primer is used to specifically detect the G allele.

2. The KASP marker primer combination for clubroot resistance in radishes according to claim 1, characterized in that, The 5′ end of the first allele-specific primer contains a universal adapter sequence corresponding to the FAM fluorescence channel, and the 5′ end of the second allele-specific primer contains a universal adapter sequence corresponding to the VIC fluorescence channel.

3. The KASP marker primer combination for clubroot resistance in radishes according to claim 2, characterized in that, The first allele-specific primer, the second allele-specific primer, and the universal reverse primer are not directly linked to fluorescent groups.

4. A KASP method for identifying genotypes related to clubroot disease resistance in radishes, characterized in that, Genotyping of the radish material to be tested using the KASP marker primer combination for clubroot resistance as described in claim 2 includes the following steps: Genomic DNA was extracted from the radish material to be tested; The genomic DNA was amplified using the aforementioned KASP marker primer combination for clubroot disease resistance in radishes; The endpoint fluorescence signal of the KASP amplification reaction was collected using the FAM and VIC fluorescence channels. Based on the clustering results of the endpoint fluorescence signal, the A / G polymorphism at the chr5:48378031 site of the radish genome was genotyped to determine whether the radish material to be tested was of AA genotype, A / G genotype or GG genotype. The FAM fluorescent channel corresponds to the A allele, and the VIC fluorescent channel corresponds to the G allele.

5. The KASP identification method for radish clubroot resistance-related genotypes according to claim 4, characterized in that, Before KASP amplification, the concentration of the genomic DNA was adjusted to 100 ng / μL, and the first allele-specific primer, the second allele-specific primer, and the universal reverse primer were prepared to 10 μM. The total reaction volume for KASP amplification was 10 μL, and the reaction system included: 5 μL of PCR premix for 2×KASP genotyping; 1 μL of the genomic DNA at a concentration of 100 ng / μL; 0.1 μL of the first allele-specific primer at a concentration of 10 μM; 0.1 μL of the second allele-specific primer at a concentration of 10 μM; 0.3 μL of the universal reverse primer at a concentration of 10 μM; And 3.5 μL of ddH2O.

6. The KASP identification method for radish clubroot resistance-related genotypes according to claim 5, characterized in that, The KASP amplification and fluorescence signal acquisition include: Pre-denaturation at 95℃ for 5 minutes; Ten cycles of specific amplification were performed, each cycle consisting of denaturation at 95°C for 15 seconds and annealing for 15 seconds, with the annealing temperature of 61°C in the first cycle and decreasing by 0.6°C in each subsequent cycle. Forty cycles of amplification were performed, each cycle consisting of denaturation at 95°C for 15 seconds and reaction at 55°C for 1 minute, and FAM fluorescence signal and VIC fluorescence signal were collected. The sample was kept at 35°C for 30 seconds, and the FAM and VIC fluorescence signals were collected at the endpoint.

7. The KASP identification method for radish clubroot resistance-related genotypes according to claim 6, characterized in that, A two-dimensional scatter plot is generated based on the FAM fluorescence signal intensity and VIC fluorescence signal intensity at the endpoint. The FAM fluorescence signal intensity is used as the Y-axis and the VIC fluorescence signal intensity is used as the X-axis. The AA genotype, A / G genotype, or GG genotype is determined based on the clustering region of the radish material to be tested in the two-dimensional scatter plot.

8. Application of KASP marker primer combination for clubroot resistance in radish in screening clubroot-resistant breeding materials, characterized in that, Molecular marker-assisted screening of radish breeding materials to be screened was performed using the KASP marker primer combination for clubroot resistance in radish as described in any one of claims 1 to 3, including the following steps: Genomic DNA was extracted from the radish breeding materials to be screened. The genomic DNA was amplified using the aforementioned KASP marker primer combination for clubroot resistance in radish, and genotyping was performed on the A / G polymorphism at the chr5:48378031 site in the radish genome. Based on the genotyping results, radish materials with the AA or A / G genotype at the chr5:48378031 locus were selected as breeding materials for radish resistance to clubroot carrying the A allele.

9. The application of the KASP marker primer combination for clubroot resistance in radish according to claim 8 in screening clubroot-resistant radish breeding materials, characterized in that, The radish breeding material to be screened is a single plant from a backcross generation population; In any backcross generation of backcross breeding, the chr5:48378031 locus of the corresponding backcross generation single plant was genotyped using the aforementioned KASP marker primer combination for clubroot resistance of radish, and A / G genotype single plants were screened as breeding materials for subsequent backcrossing or self-pollination. The A / G genotype single plants were used as breeding materials for radishes resistant to clubroot carrying the A allele.

10. The application of the KASP marker primer combination for clubroot resistance in radish according to claim 9 in screening clubroot-resistant radish breeding materials, characterized in that, The radish breeding materials to be screened also include the BC3F2 population obtained by self-pollination of single plants with the A / G genotype at chr5:48378031 locus from the BC3F1 population formed by the third backcross generation; The BC3F2 population was genotyped at the chr5:48378031 locus using the KASP marker primer combination for clubroot resistance in radishes. Individual plants with the AA genotype were selected as radish breeding materials homozygous for the A allele and used to obtain the BC3F3 generation.

Images