A SNP molecular marker, primer set, application and method for the cytoplasmic male sterility gene of Brassica napus Polima

Abstract

The present invention relates to the field of molecular biology technology, and specifically to a SNP molecular marker, primer set, application and method of the Brassica napus Polima cytoplasmic sterility gene. The SNP molecular marker is BN9000345. The primer set includes BN9000345_K01_X, BN9000345_K01_Y and BN9000345_K01_C. The application is the use of the SNP molecular marker in detecting whether Brassica napus is of the Polima sterile cytoplasmic type. The method is used to determine whether Brassica napus is of the Polima sterile cytoplasmic type. The present invention can quickly and accurately detect whether rapeseed contains the Brassica napus Polima cytoplasmic male sterility gene, and is suitable for large-scale and high-throughput Brassica napus Polima cytoplasmic male sterility gene identification (such as purity identification of sterile line and maintainer line parents) and screening.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a SNP molecular marker, a primer set, an application and a method of a cytoplasmic male sterility gene of Brassica napus Polima. Background Art

[0002] Cytoplasmic male sterility (CMS) is a maternally inherited phenomenon characterized by the inability to produce viable pollen, caused by rearrangements in the mitochondrial genome. The Polima CMS line (Pol-CMS) is internationally recognized as the first production-worthy CMS line in Brassica napus. Rapid and efficient identification of the Pol-CMS CMS gene facilitates the precise identification and selection of breeding material types, improving breeding efficiency. It also facilitates the determination of parental purity, ensuring the quality of parental material in rapeseed and enabling the utilization of hybrid vigor.

[0003] Establishing a molecular marker-based seed purity identification technology system is an important approach to improving the efficiency of seed purity identification. To date, researchers have used molecular markers such as SRAP (Sequence Related Amplified Polymorphism), RAPD (Random Amplified Polymorphic DNA), SSR (Simple Sequence Repeat), and ISSR (Inter-Simple Sequence Repeat) to identify the purity of Brassica napus hybrids, achieving good results. However, these molecular marker technologies still cannot meet the requirements of high throughput and low cost.

[0004] In recent years, competitive allele-specific PCR (KASP) has been widely used in a variety of crops due to its simplicity, repeatability, stability, accuracy, rapidity, and low cost. Studies have shown that the sterility gene (orf) on the mitochondria of different Pol-CMS is identical, and its fertility restorer gene (Rfp) is located in the nuclear genome. Although previous researchers have developed a series of molecular markers and KASP markers linked to the Pol-CMS fertility restorer gene for molecular marker-assisted breeding and hybrid purity identification in Brassica napus, these molecular markers linked to the fertility restorer gene cannot effectively distinguish between heteroplasmic isonuclear sterile lines and maintainer lines. Therefore, it is necessary to develop molecular markers and KASP markers for the identification of Pol-CMS sterility genes.

[0005] In summary, it is necessary to develop a SNP molecular marker, primer set, application and method for the Pol-CMS cytoplasmic male sterility gene in Brassica napus, so as to provide new ideas for molecular marker-assisted breeding of the Pol-CMS sterility gene and purity identification of sterile and maintainer line parents. Summary of the Invention

[0006] The present invention aims to provide a SNP molecular marker, primer set, application and method for the cytoplasmic male sterility gene of Brassica napus. The specific technical scheme is as follows:

[0007] In a first aspect, the present invention provides a SNP molecular marker of the cytoplasmic male sterility gene of Brassica napus Polima, wherein the SNP molecular marker is BN9000345, and its nucleotide sequence is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0008] In a second aspect, the present invention provides a primer set for the SNP molecular marker, comprising BN9000345_K01_X, BN9000345_K01_Y and BN9000345_K01_C;

[0009] The nucleotide sequence corresponding to the BN9000345_K01_X is shown in SEQ ID NO.3; the nucleotide sequence corresponding to the BN9000345_K01_Y is shown in SEQ ID NO.4; and the nucleotide sequence corresponding to the BN9000345_K01_C is shown in SEQ ID NO.5.

[0010] In a third aspect, the present invention provides an application of the SNP molecular marker in detecting whether Brassica napus is of the Polimar sterile cytoplasmic type.

[0011] In a fourth aspect, the present invention provides a method for detecting whether Brassica napus is of the Polimar sterile cytoplasmic type, comprising:

[0012] Step S1, extracting genomic DNA from Brassica napus;

[0013] Step S2, using the genomic DNA extracted in step S1 as a template, detecting the BN9000345 molecular marker using the primer set described in claim 2;

[0014] Step S3: determining whether the Brassica napus is a Polyma sterile cytoplasmic type according to the detected genotype.

[0015] Optionally, in step S2, FAM and HEX fluorescent linker sequences are connected to the 5′ ends of the BN9000345_K01_X and the BN9000345_K01_Y, respectively.

[0016] Optionally, in step S3, the detected genotypes include a homozygous T allele type and an A allele type; wherein, the homozygous T allele type is the Polima sterile cytoplasm type, and the detection result is displayed in red; the A allele type is the Polima fertile cytoplasm type, and the detection result is displayed in blue.

[0017] The application of the technical solution of the present invention has at least the following beneficial effects:

[0018] The present invention provides a SNP molecular marker, primer set, application, and method for the Brassica napus Polima cytoplasmic male sterility gene. By using the SNP molecular marker of the Brassica napus Polima cytoplasmic male sterility gene and the primer set and KASP technology to detect the Brassica napus Polima cytoplasmic male sterility gene, it is possible to quickly and accurately detect whether rapeseed contains the Brassica napus Polima cytoplasmic male sterility gene, thereby utilizing SNP molecular marker-assisted selection breeding. This is simple and effective and can reduce breeding costs and breeding cycles. In addition, the developed SNP molecular marker is genotyped using KASP technology. The KASP technology process performs basic automation such as DNA extraction, PCR system construction, and fluorescence signal detection, enabling high-throughput detection in 96-, 384-, and 1536-well plates. This method is suitable for large-scale and high-throughput Brassica napus Polima cytoplasmic male sterility gene identification (such as purity identification of sterile and maintainer line parents) and screening.

[0019] In addition to the above-described objects, features and advantages, the present invention has other objects, features and advantages. The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0021] Figure 1 This is a diagram of the genotyping test results of Example 1. DETAILED DESCRIPTION

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention are within the scope of protection of the present invention.

[0023] Example 1:

[0024] The method for obtaining the SNP molecular marker BN9000345 and primer set is as follows:

[0025] The upstream and downstream DNA sequences of the sterile gene (orf224) of Pol-CMS were extracted from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) and compared with the Brassica napus genome to obtain highly homologous sequences (e-value threshold of ≤2e -18 ) and there is sequence information of SNP or INDEL; SNP molecular marker primers are designed for the variable sites in the sequence information, and the primers are screened using Pol-CMS sterile lines, maintainer lines, homoplasmic restorer lines and conventional Brassica napus, and finally SNP molecular marker primers related to the Pol-CMS sterile gene are obtained, and applied to the purity identification of sterile and maintainer parents in the Polima cytoplasmic male sterility system of Brassica napus; KASP verification is performed on the sterile and maintainer parents for the candidate SNP molecular markers, and the SNP molecular marker BN9000345 with good co-segregation and amplification effect with the sterile parent is selected, and its nucleotide sequence is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0026] In the process of obtaining SNP molecular marker primers, Bacthprimer 3 software was used to design KASP primers based on the nucleotide sequences SEQ ID NO. 1 and SEQ ID NO. 2, and the primer sets with high scores (scores greater than 96 points) were screened; the primer sets were BN9000345_K01_X, BN9000345_K01_Y, and BN9000345_K01_C.

[0027] The nucleotide sequence corresponding to the BN9000345_K01_X is shown in SEQ ID NO.3; the nucleotide sequence corresponding to the BN9000345_K01_Y is shown in SEQ ID NO.4; and the nucleotide sequence corresponding to the BN9000345_K01_C is shown in SEQ ID NO.5.

[0028] The SNP molecular marker and the primer set are used to detect whether Brassica napus is of the Polimar sterile cytoplasmic type, and the detection method is as follows:

[0029] Step S1, extracting genomic DNA from Brassica napus using the CTAB (specifically cetyltrimethylammonium bromide) method;

[0030] Step S2, using the genomic DNA extracted in step S1 as a template, and detecting the BN9000345 molecular marker using the primer set described in claim 2; specifically, FAM and HEX fluorescent linker sequences are connected to the 5′ ends of the BN9000345_K01_X and the BN9000345_K01_Y, respectively; when using primers, the BN9000345_K01_X and BN9000345_K01_C are used in combination; the BN9000345_K01_Y and BN9000345_K01_C are used in combination; PCR amplification is performed using the primer set; the PCR amplification system is shown in Table 1; wherein, 2×KASP Master Mix is ​​a product of LGC Company, and the product catalog number is KBS-1016-002.

[0031] Table 1 PCR amplification system

[0032] Final concentration Actual dosage 100UMBN9000345_K01_C 0.42 μM 0.0033 μL 100UMBN9000345_K01_X 0.17 μM 0.0013 μL 100UMBN9000345_K01_Y 0.17 μM 0.0013 μL 2×KASPMasterMix 1× 0.3945 μL Ultra-pure water 0.3995 μL DNA (dry) 20 ng - 50 ng Total volume

[0033] The PCR reaction program (specifically, the Touchdown PCR reaction program) included: denaturation at 94°C for 15 min; denaturation at 95°C for 20 s, annealing and extension at 65°C-56°C for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 0.8°C each cycle; denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, 30 cycles.

[0034] At the same time, a blank control without adding template DNA to the reaction system was set up, and one blank control was set up for each PCR plate.

[0035] PCR amplification products were scanned using a PHERAstar dual-excitation plate reader with excitation wavelengths of 485 nm and emission wavelengths of 520 nm for FAM; 528 nm and 560 nm for HEX; and 575 nm and 610 nm for the system reference fluorescence ROX. Three replicates were performed for each PCR amplification product sample.

[0036] After the PCR amplification reaction is completed, the KASP reaction product is scanned using a scanner, and the fluorescence scan results are automatically converted into a graph. Kraken software is used to analyze the scan data of the PHERAstar dual-excitation plate reader.

[0037] Step S3: determining whether the Brassica napus L. is a sterile cytoplasmic type according to the detected genotype.

[0038] In Brassica napus, 58 sterile parent samples, 8 maintainer parent samples, 24 homoplasmic restorer parent samples, 2 sterile cytoplasm standards, 2 fertile cytoplasm standards and 2 blank control groups were selected into a 96-well plate, and the above detection method was used to verify the marker assay typing.

[0039] The specific results are analyzed as follows:

[0040] See also 0.8 μL The samples were genotyped into two clusters using the BN9000345 molecular marker assay: Class I genotyping and Class II genotyping. Fifty-eight sterile parent samples (excluding five undetected samples), 24 homoplasmic restorer parent samples (excluding four undetected samples), and two sterile cytoplasm standards are displayed in red on the left side of the graph and are classified as Class I genotyping, indicating that the samples contain the homozygous T allele at the KASP marker locus and their cytoplasm is Polima sterile cytoplasm. Eight maintainer parent samples and two fertile cytoplasm standards are displayed in blue on the lower right corner of the graph and are classified as Class II genotyping, indicating that the samples contain the A allele at the KASP marker locus and their cytoplasm is Polima fertile cytoplasm. Nine undetected samples and two blank controls are marked with N and displayed in gray.

[0041] Example 2:

[0042] The same method as in Example 1 was used to obtain the SNP molecular marker BN9000345 and primer set; the same detection method as in Example 1 was used. Unlike Example 1, in Brassica napus, 58 maintainer material samples (specifically samples A1-A12, B1-B12, C1-C12, D1-D12, and E1-E10), 33 sterile material samples (specifically samples F1-F12, G1-G12, and H1-H9), 2 fertile cytoplasm standard samples (specifically samples E11-E12), 2 sterile cytoplasm standard samples (specifically samples H10-H11), and 1 blank control (H12) were selected to form a 96-well plate, and the marker assay typing was verified using the above detection method.

[0043] The specific results are analyzed as follows:

[0044] Referring to Table 2, each sample was typed into two types in the BN9000345 molecular marker test, namely, the A allele type (specifically, the AA genotype or the TA genotype) and the homozygous T allele type; among them, 58 maintainer material samples (except C6, D3 and E6) and 2 fertile cytoplasm standard sample samples were all containing the A allele type, and the genotype was shown as AA or TA, which was the Polima fertile cytoplasm type; 33 sterile material samples and 2 sterile cytoplasm standard sample samples were all homozygous T allele type, and the genotype was shown as TT, which was the Polima sterile cytoplasm type.

[0045] Table 2 Genotyping test results of Example 2

[0046] 1 2 3 4 5 6 7 8 9 10 11 12 A Figure 1 A:A A:A A:A A:A A:A A:A A:A A:A A:A A:A A:A B A:A A:A A:A A:A A:A A:A A:A A:A A:A A:A A:A A:A C A:A T:A A:A A:A A:A N T:A A:A A:A T:A A:A A:A D A:A T:A N A:A A:A A:A T:A A:A A:A A:A A:A A:A E A:A A:A A:A A:A A:A N A:A A:A A:A A:A A:A A:A F A:A T:T T:T T:T T:T T:T T:T T:T T:T T:T T:T T:T G T:T T:T T:T T:T T:T T:T T:T T:T T:T T:T T:T T:T H T:T T:T T:T T:T T:T T:T T:T T:T T:T T:T T:T T:T N

[0047] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of a primer set for detecting SNP molecular markers in detecting whether Brassica napus is of the Polimar sterile cytoplasmic type, characterized in that: The SNP molecular marker is BN9000345, and its nucleotide sequence is SEQ ID NO.1 or SEQ ID NO.2; The primer set includes BN9000345_K01_X, BN9000345_K01_Y and BN9000345_K01_C; The nucleotide sequence corresponding to BN9000345_K01_X is SEQ ID NO.3; the nucleotide sequence corresponding to BN9000345_K01_Y is SEQ ID NO.4; the nucleotide sequence corresponding to BN9000345_K01_C is SEQ ID NO.5; The detected genotypes include homozygous T allele type and A allele type; A allele type includes AA genotype and TA genotype; among them, the homozygous T allele type is the Polima sterile cytoplasm type; the A allele type is the Polima fertile cytoplasm type.

2. A method for detecting whether Brassica napus is of the Polimar sterile cytoplasmic type, characterized in that: include: Step S1, extracting genomic DNA from Brassica napus; Step S2, using the genomic DNA extracted in step S1 as a template, and using the primer set as described in claim 1 to detect the BN9000345 molecular marker as described in claim 1; Step S3, determining whether the Brassica napus is a Polima sterile cytoplasmic type according to the detected genotype; The detected genotypes include homozygous T allele type and A allele type; A allele type includes AA genotype and TA genotype; among them, the homozygous T allele type is the Polima sterile cytoplasm type; the A allele type is the Polima fertile cytoplasm type.

3. The method according to claim 2, characterized in that In step S2, FAM and HEX fluorescent linker sequences were connected to the 5' ends of BN9000345_K01_X and BN9000345_K01_Y, respectively.

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