A SNP molecular marker of brassica napus polima cytoplasmic male sterility gene, primer set, application and method

Abstract

The present application relates to the technical field of molecular biology, in particular to a SNP molecular marker of Brassica napus polima cytoplasmic male sterility gene, primer group, application and method. The SNP molecular marker is BN9000344. The primer group comprises BN9000344_K01_X, BN9000344_K01_Y and BN9000344_K01_C. The application is the application of the SNP molecular marker in detecting whether Brassica napus is of polima sterile cytoplasm type. The method is used for determining whether Brassica napus is of polima sterile cytoplasm type. The present application can quickly and accurately detect whether Brassica napus contains Brassica napus polima cytoplasmic male sterility gene, and is suitable for large-scale and high-throughput identification (such as purity identification of sterile line and maintainer parent) and screening of Brassica napus polima cytoplasmic male sterility gene.

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 genotype includes a T allele-containing genotype and a homozygous C allele genotype; wherein the T allele-containing genotype is a Polima sterile cytoplasm type, and the detection result is displayed as red or purple; and the homozygous C allele genotype is a Polima fertile cytoplasm type, and the detection result is displayed as blue.

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

[0018] The SNP molecular marker, primer set, application and method of the Brassica napus Polima cytoplasmic male sterility gene provided by the application can quickly and accurately detect whether the Brassica napus Polima cytoplasmic male sterility gene is contained in the Brassica napus by detecting the Brassica napus Polima cytoplasmic male sterility gene by using the SNP molecular marker, the primer set and the KASP technology, so that the SNP molecular marker assisted selection breeding can be used, which is simple and effective, and can reduce the breeding cost and the breeding period. In addition, the SNP molecular marker is genotyped by using the KASP technology, and the basic automation such as DNA extraction, PCR system construction and fluorescence signal detection is performed in the KASP technology process, so that 96, 384 and 1536 well plates can be used for high-throughput detection, and the Brassica napus Polima cytoplasmic male sterility gene identification (such as purity identification of sterile lines and maintainer parent lines) and screening can be applied to large-scale and high-throughput.

[0019] In addition to the purposes, features and advantages described above, the application has other purposes, features and advantages. The application will be further described below with reference to the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0020] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application, illustrate the preferred embodiments of the application and assist in the explanation of the application. In the drawings:

[0021] Figure 1 is a genotyping detection result graph of Example 1;

[0022] Figure 2 is a genotyping detection result graph of sterile line material sample 24SZ06 in Example 2;

[0023] Figure 3 is a genotyping detection result graph of sterile line material sample 24SZ07 in Example 2;

[0024] Figure 4 is a genotyping detection result graph of sterile line material sample 24SZ08 in Example 2. DETAILED DESCRIPTION

[0025] With reference to the accompanying drawings, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art belong to the scope of protection of the present application.

[0026] Embodiment 1

[0027] The method for obtaining the SNP molecular marker BN9000344 and the 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 were aligned with the Brassica napus genome to obtain sequence information with a high degree of homology (e-value threshold of≤2e -18 ) and SNP; SNP molecular marker primers were designed for the variation sites in the sequence information, and the primers were screened using the Pol-CMS sterile line, the maintainer line, the homogeneous restorer line and the conventional Brassica napus, and finally the SNP molecular marker primers related to the Pol-CMS sterile gene were obtained and applied to the purity identification of the sterile line and the maintainer line parents in the Brassica napus Polima cytoplasmic male sterility system; the sterile line parent and the maintainer line parent were verified by KASP for the candidate SNP molecular marker, and the SNP molecular marker BN9000344 with good segregation and amplification effect was selected from the sterile line parent, and the nucleotide sequence thereof is as shown in SEQ ID NO. 1.

[0029] In the process of obtaining the SNP molecular marker primer, the KASP primer was designed for the nucleotide sequence SEQ ID NO. 1 by using Bacthprimer 3 software, and the primer set with a high score (score higher than 96) in the design result was screened; the primer set is BN9000344_K01_X, BN9000344_K01_Y and BN9000344_K01_C;

[0030] 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; and the nucleotide sequence corresponding to the BN9000344_K01_C is as shown in SEQ ID NO. 4.

[0031] The SNP molecular marker and the primer set are used to detect whether the Brassica napus is of the Polima sterile cytoplasmic type, and the detection method is as follows:

[0032] Step S1, genomic DNA was extracted from Brassica napus by CTAB (specifically, cetyltrimethylammonium bromide) method;

[0033] Step S2, the BN9000344 molecular marker was detected by using the primer set as claimed in claim 2 and taking the genomic DNA extracted in step S1 as a template; specifically, FAM and HEX fluorescent linker sequences were connected to the 5' ends of the BN9000344_K01_X and the BN9000344_K01_Y respectively; when the primers were used, the BN9000344_K01_X and the BN9000344_K01_C were used in combination; the BN9000344_K01_Y and the BN9000344_K01_C were used in combination; PCR amplification was performed by using the primer set; the PCR amplification system is shown in Table 1; wherein, 2xKASP Master Mix is a product of LGC company, 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, Touchdown PCR reaction program) comprises: denaturation at 94℃ for 15 min; denaturation at 95℃ for 20 s, annealing and extension at 65℃-56℃ for 60 s, 10 cycles, and the annealing and extension temperature decreases by 0.8℃ for each cycle; denaturation at 94℃ for 20 s, annealing and extension at 57℃ for 60 s, 30 cycles.

[0037] At the same time, a blank control without adding template DNA in the reaction system was set, and 1 blank control was set for each PCR plate.

[0038] The PCR amplification products were scanned by a bidirectional single excitation plate reader PHERAstar, the FAM excitation wavelength was 485 nm, and the emission wavelength was 520 nm; the HEX excitation wavelength was 528 nm, and the emission wavelength was 560 nm; the system reference fluorescence ROX excitation wavelength was 575 nm, and the emission wavelength was 610 nm. Each PCR amplification product sample was set with 3 repeats.

[0039] After the completion of the PCR amplification reaction, the KASP reaction product was read by a scanner, and the fluorescence scanning results were automatically converted into a graph. The PHERAstar scanning data was analyzed by using Kraken software.

[0040] Step S3, whether the Brassica napus is of the Polima sterile cytoplasm type was determined 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 samples, 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 are intended to be within the scope of protection of the present invention.

Claims

1. Use of a primer set for SNP molecular markers in detecting whether Brassica napus is of the Polimar sterile cytoplasmic type, characterized in that: The SNP molecular marker is BN9000344, and its nucleotide sequence is as SEQ ID NO.1; The primer set includes BN9000344_K01_X, BN9000344_K01_Y and BN9000344_K01_C; The nucleotide sequence corresponding to BN9000344_K01_X is shown in SEQ ID NO.2; the nucleotide sequence corresponding to BN9000344_K01_Y is shown in SEQ ID NO.3; the nucleotide sequence corresponding to BN9000344_K01_C is shown in SEQ ID NO.4; The detected genotypes include T allele type and homozygous C allele type; among them, the T allele type is the sterile cytoplasm type of Polima; the homozygous C allele type is the fertile cytoplasm type of Polima.

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, detecting the BN9000344 molecular marker using the primer set described in claim 1; Step S3: determining whether the Brassica napus is a Polyma sterile cytoplasmic type according to the detected genotype.

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 BN9000344_K01_X and BN9000344_K01_Y, respectively.

4. The method according to claim 3, 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