Application and method of primer group of SNP molecular marker in detecting whether cabbage type rape is radish sterile cytoplasm type
By developing the SNP molecular marker primer set BN900568_K01, the low throughput and high cost problems of cytoplasmic type detection in Brassica napus radish sterility were solved, achieving rapid and efficient cytoplasmic type identification, improving breeding efficiency and accuracy, and making it suitable for high-throughput screening in the rapeseed industry.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 湖南省作物研究所
- Filing Date
- 2026-01-20
- Publication Date
- 2026-04-14
AI Technical Summary
Existing technologies suffer from low throughput, high cost, and low efficiency when detecting cytoplasmic male sterility types in Brassica napus, making it difficult to meet the breeding needs of high throughput, low cost, and precision. Furthermore, the detection of cytoplasmic male sterility genes is lagging and cannot be screened simultaneously with nuclear genes.
A primer set BN900568_K01 for SNP molecular markers was developed, including primers BN9000568_K01_X, BN9000568_K01_Y, and BN9000568_K01_C. This primer set enables rapid, high-throughput detection of cytoplasmic sterility types in radishes using the KASP detection system. Combined with FAM and HEX fluorescent adapter sequences, it can detect 384 samples in a single reaction, providing accurate results at a low cost.
It achieves high-throughput, low-cost detection of cytoplasmic types of radish sterility with a false positive rate of less than 0.1%, is compatible with restorer gene markers, significantly improves breeding efficiency, is applicable to sterile line propagation and restorer line selection, and meets the high-throughput screening needs of the rapeseed seed industry.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, and in particular to the application and method of a primer set of SNP molecular markers in detecting whether Brassica napus is a radish-type sterile cytoplasmic type. Background Technology
[0002] Hybrid vigor can be utilized to improve rapeseed yield, resistance, and oil content. Cytoplasmic male sterility (CMS) systems, due to their stable sterility, wide range of restorer sources, and high seed purity, have become the core technical approach for rapeseed hybrid seed production. Among various CMS types, Ogu CMS, derived from radish (Raphanus sativus L.), is hailed as the "golden sterility source" because of its complete sterility and its independence from genetic background and environmental conditions.
[0003] However, the large-scale application of Ogu CMS in breeding practice still faces two major bottlenecks: First, the reproduction of sterile lines and hybrid seed production are prone to contamination of restoration genes or cytoplasmic mixing, leading to a decrease in seed purity; second, traditional testcross identification requires a complete growth cycle, which is time-consuming, costly, and inefficient, making it difficult to meet the current upgrading needs of the rapeseed seed industry for "high throughput, low cost, and precision." Previous studies have conducted extensive research on the molecular genetic basis of Ogu CMS, clarifying that its sterility is caused by the mitochondrial chimeric gene orf138, while fertility restoration is mediated by the nuclear gene Rfo (also known as Rfp). In recent years, researchers have used markers such as SSR, SRAP, RAPD, and ISSR to conduct tight linkage screening of nuclear genes Rfo. Based on this, codominant SNP-KASP markers BN900009_K02, BN9000018_K01, BN9000019_K01+BN9000020_K01, and BN9000019_K01+BN9000020_K02 have been developed, which can achieve high-throughput detection and are suitable for large-scale, high-throughput identification and screening of cytoplasmic sterility restoration genes in Brassica napus and radish.
[0004] However, the development of cytoplasmic sterility genes lags significantly behind that of nuclear fertility restoration genes (Rfo). Currently, production still relies on the SCAR marker ORF138-F / R, designed for orf138, for conventional PCR-agarose electrophoresis. While this assay is highly specific and stable, it suffers from low throughput, significant human error, and difficulty in automated data acquisition, severely limiting its efficiency in simultaneous screening with nuclear gene Rfo markers.
[0005] Therefore, developing high-throughput, low-cost novel markers for the detection of orf138, and integrating sterility genes, fertility restoration genes, and potential fertility modification sites into a single KASP detection system, has become a critical technical bottleneck that needs to be overcome in Ogu CMS molecular breeding. Thus, it is necessary to provide an application and method for a primer set of SNP molecular markers in detecting whether Brassica napus has a radish-like sterile cytoplasmic type, to achieve rapid, high-throughput detection of Ogu CMS sterile cytoplasm. Summary of the Invention
[0006] The purpose of this invention is to provide an application and method for using SNP molecular marker primer sets in detecting whether Brassica napus is a radish-type sterile cytoplasmic type. The specific technical solution is as follows: In a first aspect, the present invention provides an application of a primer set of an SNP molecular marker in detecting whether Brassica napus is a radish-type sterile cytoplasmic type, wherein the SNP molecular marker is BN900568_K01 and its nucleotide sequence is SEQ ID NO.1 or SEQ ID NO.2; The primer set includes BN9000568_K01_X, BN9000568_K01_Y and BN9000568_K01_C; The nucleotide sequence corresponding to BN9000568_K01_X is SEQ ID NO.3; The nucleotide sequence corresponding to BN9000568_K01_Y is SEQ ID NO.4; The nucleotide sequence corresponding to BN9000568_K01_C is SEQ ID NO.5; The detected genotypes included homozygous C genotypes and C-deleted genotypes; among them, the C-deleted genotype was the radish-derived sterile cytoplasmic type, and the homozygous C genotype was the radish-derived fertile cytoplasmic type.
[0007] In a second aspect, the present invention provides a method for detecting whether Brassica napus is of the radish-type sterile cytoplasmic type, comprising: Step S1: Extract genomic DNA from Brassica napus; Step S2: Using the genomic DNA extracted in step S1 as a template, the BN900568_K01 molecular marker described in claim 1 is detected using the primer set described in claim 1. Step S3: Determine whether the Brassica napus is a radish-type sterile cytoplasmic type based on the detected genotype.
[0008] Optionally, in step S2, FAM and HEX fluorescent connector sequences are connected to the 5' ends of BN9000568_K01_X and BN9000568_K01_Y, respectively.
[0009] Optionally, the detected genotypes include homozygous C genotypes and C-deleted genotypes; wherein, the C-deleted genotype is a radish-type sterile cytoplasmic type; and the homozygous C genotype is a radish-type fertile cytoplasmic type.
[0010] Compared with existing SCAR markings, the technical solution of this invention has at least the following advantages: (1) High throughput: A single reaction can complete the typing of 384 samples and obtain the test results within 3 hours, meeting the early screening needs of large-scale breeding materials; (2) The results are accurate and reproducible: the SNP site is specifically located inside orf138, and there is no cross-amplification with normal rapeseed cytoplasm, with a false positive rate of less than 0.1%; (3) Low cost: No agarose gel electrophoresis is required, eliminating the need for restriction endonucleases and fluorescent probes, reducing the cost of single-sample detection by more than 60% compared to SCAR labeling; (4) Compatible with the restorer gene Rfo-KASP marker: The SNP marker of the radish sterility gene in Brassica napus in this invention can be simultaneously detected with the reported restorer gene markers BN9000018_K01, BN9000019_K01+BN9000020_K01, and BN9000019_K01+BN9000020_K02 under the same PCR program and fluorescence channel, realizing dual high-throughput screening of "sterile cytoplasm + restorer gene", which significantly improves the efficiency of Ogu CMS three-line breeding; (5) Wide range of applications: It can be used for monitoring cytoplasmic purity during the breeding process of sterile lines, early prospect selection of restorer line backcrossing and conversion, and sampling inspection of commercial seed quality, providing standardized and automated molecular detection tools for rapeseed hybrid seed industry.
[0011] In summary, this invention fills the gap in high-throughput SNP markers for the Ogu CMS sterility gene, constructs a KASP detection system that runs parallel to the restorer gene marker, and provides a novel technical solution for Ogu CMS molecular marker-assisted breeding and parental purity identification in Brassica napus. It has significant technological advancements and industrial application value.
[0012] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the figures. Attached Figure Description
[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.
[0014] Figure 1 This is a graph showing the genotyping results of Example 1. Detailed Implementation
[0015] The technical solutions of 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 a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0016] Example 1: The method for obtaining the SNP molecular marker BN900568_K01 and primer set is as follows: DNA sequences upstream and downstream of the sterility gene (orf138) of Ogu CMS were extracted from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) and compared with the genome of Brassica napus to obtain highly homologous sequences (e-value threshold of ≤ 2e). -18 The sequence information of SNPs or INDELs is present. SNP molecular marker primers are designed for the variant sites in the sequence information. The primers are screened using Ogu CMS sterile lines, maintainer lines, homogeneous restorer lines and conventional Brassica napus. Finally, SNP molecular marker primers related to the Ogu CMS sterility gene are obtained and applied to the purity identification of sterile lines and maintainer lines in the Brassica napus radish cytoplasmic sterility system. For the candidate SNP molecular markers, KASP verification is performed on the sterile line parents and maintainer line parents. The SNP molecular marker BN900568_K01 that can identify sterile lines and maintainer lines is selected. Its nucleotide sequence is SEQ ID NO.1 or SEQ ID NO.2.
[0017] In the process of obtaining SNP molecular marker primers, Bacthprimer 3 software was used to design KASP primers for nucleotide sequences SEQ ID NO.1 or SEQ ID NO.2, and primer sets with high scores (scores higher than 96 points) were screened in the design results; the primer sets were BN9000568_K01_X, BN9000568_K01_Y and BN9000568_K01_C; The nucleotide sequence corresponding to BN9000568_K01_X is SEQ ID NO.3; The nucleotide sequence corresponding to BN9000568_K01_Y is SEQ ID NO.4; The nucleotide sequence corresponding to BN9000568_K01_C is SEQ ID NO.5.
[0018] The SNP molecular markers and primer set were used to detect whether Brassica napus was of the radish-type sterile cytoplasmic type. The detection method is as follows: Step S1: Genomic DNA was extracted from Brassica napus using the CTAB (Cetyltrimethylammonium bromide) method. Step S2: Using the genomic DNA extracted in Step S1 as a template, the BN9000568_K01 molecular marker is detected using the primer set. Specifically, FAM and HEX fluorescent adapter sequences are ligated to the 5' ends of BN9000568_K01_X and BN9000568_K01_Y, respectively. When using primers, BN9000568_K01_X and BN9000568_K01_C are used in combination; BN9000568_K01Y and BN9000568_K01_C are used in combination. PCR amplification is performed using the primer set. The PCR amplification system is shown in Table 1. Among them, 2×KASP Master Mix is a product of LGC Corporation, product catalog number KBS-1016-002.
[0019] Table 1 PCR amplification system The PCR reaction program (specifically the Touchdown PCR reaction program) includes: denaturation at 94℃ for 15 min; denaturation at 95℃ for 20 s, annealing and extension at 65℃-56℃ for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 0.8℃ per cycle; denaturation at 94℃ for 20 s, annealing and extension at 57℃ for 60 s, 30 cycles.
[0020] Meanwhile, a blank control was set up in the reaction system without template DNA, with one blank control set up for each PCR plate.
[0021] PCR amplification products were scanned using a two-way single-excitation plate reader, Pherastar. The excitation wavelength for FAM was 485 nm, and the emission wavelength was 520 nm; the excitation wavelength for HEX was 528 nm, and the emission wavelength was 560 nm; the system reference fluorescence ROX was excited at 575 nm and emitted at 610 nm. Each PCR amplification product sample was replicated in triplicate.
[0022] After the PCR amplification reaction was completed, the KASP reaction products were scanned using a scanner to read the fluorescence data, and the results of the fluorescence scans were automatically converted into images. The scan data from the Pherastar two-way single-excitation plate reader were analyzed using Kraken software.
[0023] Step S3: Determine whether the Brassica napus is a radish-type sterile cytoplasmic type based on the detected genotype.
[0024] In Brassica napus, 45 Ogu CMS sterile parental samples, 50 Ogu CMS maintainer parental samples, and 1 blank control group were selected to form a 96-well plate, and the above detection method was used to verify the labeling Assay genotyping.
[0025] The specific results are analyzed as follows: See Figure 1 In the BN9000568_K01 molecular marker detection, all samples were classified into two clusters: Category I genotype and Category II genotype. Among them, 45 Ogu CMS sterile parental samples were shown in blue, located in the lower right corner of the graph, and were classified as Category I genotype. This classification indicates that the samples lack the C genotype at this KASP marker site, and their cytoplasm is of the radish-like sterile cytoplasmic type. The 50 Ogu CMS maintainer parental samples were shown in red, located in the upper left corner of the graph, and were classified as Category II genotype. This classification indicates that the samples contain the homozygous C genotype at this KASP marker site, and their cytoplasm is of the radish-like fertile cytoplasmic type. One blank control was marked with N, indicating no detection, and was shown in gray.
[0026] Example 2: The same method as in Example 1 was used to obtain the SNP molecular marker BN9000568_K01 and primer set; the same detection method as in Example 1 was also used. The difference from Example 1 is that, in Brassica napus, 24 Ogu CMS male-sterile line samples, 24 Ogu CMS maintainer line samples, 24 Ogu CMS homologous restorer line samples, 12 Pol CMS (Polima cytoplasmic male sterility) male-sterile line samples, 10 conventional rapeseed inbred line samples, and 2 blank control samples were assembled into a 96-well plate, and the marker Assay genotyping was verified using the above detection method.
[0027] The specific results are analyzed as follows: Referring to Table 2, the samples were classified into two clusters in the BN9000568_K01 molecular marker detection: genotype I and genotype II. The 24 Ogu CMS male-sterile lines and 24 Ogu CMS homozygous restorer lines were all shown in blue, classified as genotype I, indicating a deficiency of the C genotype at this KASP marker site, with radish-like male-sterile cytoplasm. The 24 Ogu CMS maintainer lines, 12 Pol CMS male-sterile lines, and 10 conventional rapeseed inbred lines were all shown in red, classified as genotype II, indicating the presence of a homozygous C genotype at this KASP marker site, with radish-like fertile cytoplasm. Two blank controls were marked with N, indicating no detection.
[0028] Table 2. Genotyping results of Example 2 The above description is only a preferred embodiment of the present invention and does not limit the scope of the present invention. All equivalent structural transformations made under the inventive concept of the present invention using the contents of the present invention specification and drawings, or direct / indirect applications in other related technical fields, are included within the protection scope of the present invention.
Claims
1. The application of SNP molecular marker primer sets in detecting whether Brassica napus is a radish-type sterile cytoplasmic type, characterized in that, The SNP molecular marker is BN900568_K01, and its nucleotide sequence is SEQ ID NO.1 or SEQ ID NO.2; The primer set includes BN9000568_K01_X, BN9000568_K01_Y and BN9000568_K01_C; The nucleotide sequence corresponding to BN9000568_K01_X is SEQ ID NO.3; The nucleotide sequence corresponding to BN9000568_K01_Y is SEQ ID NO.4; The nucleotide sequence corresponding to BN9000568_K01_C is SEQ ID NO.5; The detected genotypes included homozygous C genotypes and C-deleted genotypes; among them, the C-deleted genotype was the radish-derived sterile cytoplasmic type, and the homozygous C genotype was the radish-derived fertile cytoplasmic type.
2. A method for detecting whether Brassica napus is of the radish-type sterile cytoplasmic type, characterized in that, include: Step S1: Extract genomic DNA from Brassica napus; Step S2: Using the genomic DNA extracted in step S1 as a template, the BN900568_K01 molecular marker described in claim 1 is detected using the primer set described in claim 1. Step S3: Determine whether the Brassica napus is a radish-type sterile cytoplasmic type based on the detected genotype.
3. The method for detecting whether Brassica napus is of the radish-type sterile cytoplasmic type as described in claim 2, characterized in that, In step S2, FAM and HEX fluorescent connector sequences are connected to the 5' ends of BN9000568_K01_X and BN9000568_K01_Y, respectively.
4. The method for detecting whether Brassica napus is of the radish-type sterile cytoplasmic type as described in claim 2, characterized in that, The detected genotypes included homozygous C genotypes and C-deleted genotypes; among them, the C-deleted genotype was the radish-derived sterile cytoplasmic type, and the homozygous C genotype was the radish-derived fertile cytoplasmic type.