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 BN9000344. The primer group comprises a primer group BN9000344K01X, a primer group BN9000344K01Y and a primer group BN9000344K01C, and the primer group comprises a primer group BN9000344K01X, a primer 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 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 SNP molecular markers, primer sets, applications and methods for the cytoplasmic male sterility gene of Brassica napus. The specific technical solutions are 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 BN9000344, and its nucleotide sequence is shown in SEQ ID NO.1.

[0008] In a second aspect, the present invention provides a primer set for SNP molecular markers, comprising BN9000344_K01_X, BN9000344_K01_Y and BN9000344_K01_C;

[0009] The nucleotide sequence corresponding to the BN9000344_K01_X is shown in SEQ ID NO.2; the nucleotide sequence corresponding to the BN9000344_K01_Y is shown in SEQ ID NO.3; and the nucleotide sequence corresponding to the BN9000344_K01_C is shown in SEQ ID NO.4.

[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 BN9000344 molecular marker using the primer set;

[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 BN9000344_K01_X and the BN9000344_K01_Y, respectively.

[0016] Optionally, in step S3, the detected genotypes include a T allele type and a homozygous C allele type; wherein, the T allele type is the Polima sterile cytoplasm type, and the detection result is displayed in red or purple; the homozygous C 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 is a diagram of the genotyping test results of Example 1;

[0022] Figure 2 This is a diagram showing the genotyping results of the sterile material sample 24SZ06 in Example 2;

[0023] Figure 3 This is a diagram showing the genotyping results of the sterile material sample 24SZ07 in Example 2;

[0024] Figure 4 This is a diagram showing the genotyping results of the sterile material sample 24SZ08 in Example 2. DETAILED DESCRIPTION

[0025] 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.

[0026] Example 1:

[0027] The method for obtaining the SNP molecular marker BN9000344 and primer set is as follows:

[0028] 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; SNP molecular marker primers were designed for the variant sites in the sequence information, and the primers were 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 were obtained, and applied to the purity identification of sterile and maintainer parents in the Polima cytoplasmic male sterility system of Brassica napus; KASP verification was performed on the sterile and maintainer parents for the candidate SNP molecular markers, and the SNP molecular marker BN9000344 with good co-segregation and amplification effect with the sterile parent was selected, and its nucleotide sequence is shown in SEQ ID NO.1.

[0029] In the process of obtaining SNP molecular marker primers, Bacthprimer 3 software was used to design KASP primers based on the nucleotide sequence SEQ ID NO. 1, and the primer sets with high scores (scores greater than 96 points) were screened; the primer sets were BN9000344_K01_X, BN9000344_K01_Y, and BN9000344_K01_C;

[0030] The nucleotide sequence corresponding to the BN9000344_K01_X is shown in SEQ ID NO.2; the nucleotide sequence corresponding to the BN9000344_K01_Y is shown in SEQ ID NO.3; and the nucleotide sequence corresponding to the BN9000344_K01_C is shown in SEQ ID NO.4.

[0031] 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:

[0032] Step S1, extracting genomic DNA from Brassica napus using the CTAB (specifically cetyltrimethylammonium bromide) method;

[0033] Step S2: Using the genomic DNA extracted in step S1 as a template, the BN9000344 molecular marker is detected using the primer set described in claim 2; specifically, FAM and HEX fluorescent linker sequences are connected to the 5′ ends of the BN9000344_K01_X and the BN9000344_K01_Y, respectively; when using primers, the BN9000344_K01_X and BN9000344_K01_C are used in combination; the BN9000344_K01_Y and BN9000344_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, and the product catalog number is KBS-1016-002.

[0034] Table 1 PCR amplification system

[0035] Final concentration Actual usage 100UMBN9000344_K01_C 0.42μM 0.0033μL 100UMBN9000344_K01_X 0.17 μM 0.0013μL 100UMBN9000344_K01_Y 0.17 μM 0.0013μL 2×KASPMasterMix 1× 0.3945μL Ultrapure water 0.3995μL DNA (dried) 20ng-50ng Total volume 0.8μL

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] Step S3: determining whether the Brassica napus is a Polyma sterile cytoplasmic type according to the detected genotype.

[0041] In Brassica napus, 42 sterile parent samples, 24 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.

[0042] The specific results are analyzed as follows:

[0043] See also Figure 1 The samples were genotyped into two clusters using the BN9000344 molecular marker assay: Class I genotyping and Class II genotyping. The 42 sterile parent samples (excluding one undetected sample), 24 homoplasmic restorer parent samples, and two sterile cytoplasm standards are all displayed in purple at the top of the graph and are classified as Class I genotyping, indicating that the samples contain the T allele at the KASP marker locus, representing the Polima sterile cytoplasm type. The 24 maintainer parent samples and two fertile cytoplasm standards are all displayed in blue at the bottom right corner of the graph and are classified as Class II genotyping, indicating that the samples contain the homozygous C allele at the KASP marker locus, representing the Polima fertile cytoplasm type. One undetected sample and two blank controls are displayed in gray.

[0044] Example 2:

[0045] The same method as in Example 1 was used to obtain the SNP molecular marker BN9000344 and primer set; the same detection method as in Example 1 was used. Unlike Example 1, three groups of sterile material samples, 24SZ06, 24SZ07, and 24SZ08, were selected from Brassica napus. The marker assay typing results were verified using the above-described detection method for these three groups of sterile material samples. Two 96-well plates, totaling 192 samples, were selected from each group of sterile material samples. One sterile cytoplasm standard sample and one fertile cytoplasm standard sample were placed in each 96-well plate as control samples.

[0046] The specific results are analyzed as follows:

[0047] See Table 2 and Figure 2-Figure 4 , the three groups of sterile material samples can be typed normally, and the sterility is 100%. Specifically, in the control sample, the genotype of the sterile cytoplasm standard sample is TT or TC. Figure 2-Figure 4 In the figure, the color is red (representing the genotype of TT) or purple (representing the genotype of TC); the genotype of the fertile cytoplasm standard is CC. Figure 2-Figure 4 The genotypes of the three groups of sterile material samples 24SZ06, 24SZ07 and 24SZ08 to be tested were all shown as TT or TC. Figure 2-Figure 4The color in the middle is red (representing the genotype is TT) or purple (representing the genotype is TC), indicating that the three groups of sterile material samples can be typed normally and the sterility is 100%.

[0048] Table 2 Genotyping test results of Example 2

[0049] Identification number Sterile cytoplasm standard Fertile cytoplasm standard Samples of sterile material to be tested 24SZ06 TC CC / N TC / TT 24SZ07 TC CC / N TC / TT 24SZ08 TC CC / CC TC / TT

[0050] 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 shall be included within the scope of protection 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 BN9000344, and its nucleotide sequence is as SEQ ID NO.

1.

2. A primer set for SNP molecular markers as claimed in claim 1, characterized in that: Includes BN9000344_K01_X, BN9000344_K01_Y and BN9000344_K01_C; The nucleotide sequence corresponding to the BN9000344_K01_X is as shown in SEQ ID NO.2; the nucleotide sequence corresponding to the BN9000344_K01_Y is as shown in SEQ ID NO.3; the nucleotide sequence corresponding to the BN9000344_K01_C is as shown in SEQ ID NO.

4.

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 BN9000344 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 BN9000344_K01_X and the BN9000344_K01_Y, respectively.

6. The method according to claim 5, characterized in that In step S3, the detected genotypes include a T allele type and a homozygous C allele type; wherein, the T allele type is the Polima sterile cytoplasm type, and the detection result is displayed in red or purple; the homozygous C allele type is the Polima fertile cytoplasm type, and the detection result is displayed in blue.

Images