SNP (Single Nucleotide Polymorphism) molecular marker, primer group, application and method for Brassica napus Bolima cytoplasmic male sterility gene

Abstract

The invention relates to the technical field of molecular biology, in particular to an SNP (Single Nucleotide Polymorphism) molecular marker of a Brassica napus Bolima cytoplasm sterile gene, a primer group, application and a method. The SNP (Single Nucleotide Polymorphism) molecular marker is BN9000345. The primer group comprises a primer group BN9000345K01X, a primer group BN9000345K01Y and a primer group BN9000345K01C, and the primer group comprises a primer group BN9000345K01X, a primer group The application refers to the application of the SNP molecular marker in detecting whether the brassica napus is of the Brassica napus sterile cytoplasm type or not. The method is used for determining whether the brassica napus is of the Brassica napus sterile cytoplasm type or not. The method can rapidly and accurately detect whether the brassica napus contains the Brassica napus Bolima cytoplasmic male sterility gene or not, and is suitable for large-scale and high-throughput Brassica napus Bolima cytoplasmic male sterility gene identification (such as purity identification of sterile line and maintainer line parents) and screening.

Description

Technical Field

[0001] The 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 Brassica napus Polima cytoplasmic sterility gene. Background Art

[0002] Cytoplasmic male sterility (CMS) is a maternal genetic phenomenon that results in the inability to produce viable pollen, caused by rearrangements in the mitochondrial genome. Polima CMS (Pol-CMS) is internationally recognized as the first cytoplasmic male sterility type of Brassica napus with production value. Rapid and efficient identification of the Pol-CMS cytoplasmic sterility gene is conducive to the accurate identification and selection of breeding material types, improving breeding efficiency; it is also conducive to identifying the purity of parents, ensuring the quality of rapeseed parent materials, and ensuring the utilization of hybrid vigor.

[0003] Establishing a seed purity identification technology system based on molecular markers is an important way to improve the efficiency of seed purity identification. So far, predecessors 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 and achieved good results. However, the above molecular marker technology cannot meet the needs of high throughput and low cost.

[0004] In recent years, competitive allele-specific PCR (Kompetitive Allele Specific PCR, KASP) has been widely used in a variety of crops due to its advantages such as simplicity, repeatability, stability, accuracy, rapidity, and low cost. Studies have shown that the sterility gene (orf) on the mitochondria of different Pol-CMS is the same, and its fertility restorer gene (Rfp) is located in the nuclear genome. Although a series of molecular markers and KASP markers linked to the fertility restorer gene of Pol-CMS have been developed by predecessors and applied to molecular marker-assisted breeding of Brassica napus and purity identification of hybrids, these molecular markers linked to the fertility restorer gene cannot effectively distinguish heteroplasmic homonuclear 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 lines 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 Polima. The specific technical scheme is as follows:

[0007] In a first aspect, the present invention provides a SNP molecular marker of the Brassica napus Polima cytoplasmic male sterility gene, wherein the SNP molecular marker is BN9000345, and its nucleotide sequence is as 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 as shown in SEQ ID NO.3; the nucleotide sequence corresponding to the BN9000345_K01_Y is as shown in SEQ ID NO.4; the nucleotide sequence corresponding to the BN9000345_K01_C is as 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 Polyma sterile cytoplasmic type.

[0011] In a fourth aspect, the present invention provides a method for detecting whether Brassica napus is of the Polyma 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, and using the primer set as described in claim 2 to detect the BN9000345 molecular marker;

[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 of a male sterile gene of Brassica napus Polima cytoplasm. By detecting the male sterile gene of Brassica napus Polima cytoplasm using the primer set and KASP technology, the SNP molecular marker of the male sterile gene of Brassica napus Polima cytoplasm can be used to quickly and accurately detect whether the Brassica napus Polima cytoplasm male sterile gene is contained in the rapeseed, thereby using the SNP molecular marker to assist in selection breeding, which is simple and effective and can reduce the breeding cost and breeding cycle. In addition, the developed SNP molecular marker is genotyped using the KASP technology, and basic automation such as DNA extraction, PCR system construction and fluorescence signal detection are performed in the KASP technology process, so that high-throughput detection of 96, 384 and 1536 well plates can be realized, which is suitable for large-scale and high-throughput identification (such as purity identification of sterile line and maintenance line parents) and screening of the male sterile gene of Brassica napus Polima cytoplasm.

[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 with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0023] Embodiment 1:

[0024] The method for obtaining the SNP molecular marker BN9000345 and the 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 genome of Brassica napus 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, homogeneous 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 the sterile line and maintainer line parents in the Brassica napus Polima cytoplasmic sterile system; for the candidate SNP molecular markers, KASP verification is performed on the sterile line parents and the maintainer line parents, and the SNP molecular marker BN9000345 with good co-segregation and amplification effect with the sterile line parents is selected, and its nucleotide sequence is as 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 for the nucleotide sequences SEQ ID NO.1 and SEQ ID NO.2, and a primer set with a high score (a score higher than 96 points) in the design results was screened; the primer set was BN9000345_K01_X, BN9000345_K01_Y and BN9000345_K01_C;

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

[0028] The SNP molecular marker and the primer set are used to detect whether the Brassica napus is of the Polyma 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, connecting FAM and HEX fluorescent linker sequences 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×KASPMaster Mix is ​​a product of LGC, 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×KASP Master Mix 1× 0.3945 μL Ultra-pure water 0.3995 μL DNA (dry) 20 ng - 50 ng Total volume 0.8 μL

[0033] The PCR reaction program (specifically Touchdown PCR reaction program) includes: 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 in 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] The PCR amplification products were scanned using the bidirectional single excitation plate reader PHERAstar, with FAM excitation wavelength of 485 nm and emission wavelength of 520 nm; HEX excitation wavelength of 528 nm and emission wavelength of 560 nm; system reference fluorescence ROX excitation wavelength of 575 nm and emission wavelength of 610 nm. Three replicates were set for each PCR amplification product sample.

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

[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 homogeneous restorer parent samples, 2 sterile cytoplasm standard samples, 2 fertile cytoplasm standard samples 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 Figure 1 , each sample was typed into two clusters in the BN9000345 molecular marker detection, namely, Class I genotyping and Class II genotyping; among them, 58 sterile parent samples (except 5 undetected samples), 24 homogeneous restorer parent samples (except 4 undetected samples) and 2 sterile cytoplasm standard samples were all displayed in red, located on the left side of the figure, and were classified as Class I genotyping, which indicated that the samples contained homozygous T allele genotype at this KASP marker site, and their cytoplasm was Polima sterile cytoplasm; 8 maintainer parent samples and 2 fertile cytoplasm standard samples were all displayed in blue, located in the lower right corner of the figure, and were classified as Class II genotyping, which indicated that the samples contained A allele genotype at this KASP marker site, and their cytoplasm was Polima fertile cytoplasm. 9 undetected samples and 2 blank controls were marked as N and displayed in gray.

[0041] Embodiment 2:

[0042] The same method as in Example 1 was used to obtain the SNP molecular marker BN9000345 and the primer set; the same detection method as in Example 1 was used. Different from 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 above detection method was used to verify the marker Assay typing.

[0043] The specific results are analyzed as follows:

[0044] Referring to Table 2, each sample was classified into two types in the BN9000345 molecular marker detection, namely, the A allele type (specifically, the AA genotype or the TA genotype) and the homozygous T allele type; among them, 58 maintenance line 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 line 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 A:A 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 T:A A:A A:A A:A T:A N A:A A:A T:A A:A A:A A:A D T:A A:A N A:A A:A T:A A: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 T:T 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 N

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A SNP molecular marker for the cytoplasmic male sterility gene of Brassica napus Polima, characterized in that: The SNP molecular marker is BN9000345, and its nucleotide sequence is shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. A primer set for SNP molecular markers as claimed in claim 1, characterized in that: Includes BN9000345_K01_X, BN9000345_K01_Y and BN9000345_K01_C; The nucleotide sequence corresponding to the BN9000345_K01_X is as shown in SEQ ID NO.3; the nucleotide sequence corresponding to the BN9000345_K01_Y is as shown in SEQ ID NO.4; the nucleotide sequence corresponding to the BN9000345_K01_C is as shown in SEQ ID NO.

5.

3. Use of the SNP molecular marker as claimed in claim 1 in detecting whether Brassica napus is of the Polyma sterile cytoplasmic type.

4. A method for detecting whether Brassica napus is of the Polyma 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 2 to detect the BN9000345 molecular marker; Step S3: determining whether the Brassica napus is a Polyma sterile cytoplasmic type according to the detected genotype.

5. The method according to claim 4, characterized in that 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.

6. The method according to claim 5, characterized in that 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 cytoplasmic type, and the detection result is displayed in red; the A allele type is the Polima fertile cytoplasmic type, and the detection result is displayed in blue.

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