Molecular marker for specifically detecting cytoplasm restoring line 206R of brassica napus radish and application of molecular marker

Abstract

The invention discloses a molecular marker for specific detection of a brassica napus radish cytoplasm restoring line 206R and application thereof, the molecular marker comprises two SNP molecular markers, the SNP sites are located at the 26276606th site and the 26190259th site on an A10 chromosome, a double-SNP marker combination is developed for the brassica napus radish cytoplasm male sterility restoring line 206R for the first time, and the molecular marker is used for specific detection of the brassica napus radish cytoplasm male sterility restoring line 206R. The technical blank that the restorer line lacks specific molecular markers is filled, and the molecular marker can be used for distinguishing whether restorer gene loci exist or not and the pure and heterozygous states in a single offspring plant of the restorer line and assisting in breeding selection.

Description

Technical Field

[0001] The invention belongs to the technical field of rapeseed breeding and molecular identification, and in particular relates to a set of molecular markers for specifically detecting a cytoplasmic restorer line 206R of cabbage-type rapeseed and radish and an application thereof. Background Art

[0002] Brassica napus has obvious hybrid vigor. Compared with conventional varieties, the use of rapeseed hybrids can increase yield, enhance adaptability and improve stress resistance. In the process of hybrid breeding, the purity of hybrids will directly affect the quality and quality of the variety. At present, the main ways to produce rapeseed hybrids in my country are cytoplasmic male sterility (CMS), nuclear male sterility (GMS), chemical male sterility (CHA) and self-incompatibility (SI). Among them, cytoplasmic male sterility is the most widely used and effective way to utilize hybrid vigor in rapeseed. The types of cytoplasmic male sterility that have been discovered or created include Polima cytoplasmic male sterility, Shaan 2A cytoplasmic male sterility, radish cytoplasmic male sterility, Nap cytoplasmic male sterility, etc.

[0003] The ogura cytoplasmic sterile line derived from radish is an ideal male sterile type for rapeseed due to its stable sterility. The French National Academy of Agricultural Sciences created the restorer line R2000 of radish cytoplasm by irradiation induction, making this sterile line widely used in Europe. However, due to the limitation of the restorer patent, the use of this sterile line in the breeding of Brassica napus was seldom in China in the early stage. In the previous research, through the method of distant hybridization combined with backcrossing, we successfully bred a new restorer line 206R (CN113016606A) of radish cytoplasm for Brassica napus. Compared with the French R2000, this restorer line has a shorter radish chromosome fragment and is located in the A10 linkage group of Brassica napus. It has a series of advantages such as good stability of intergenerational transmission, sufficient pollen, good recovery rate, normal seed setting, etc., which can be directly applied to the research on hybrid vigor breeding of rapeseed in my country.

[0004] In the breeding process, rapid breeding and identification of pure lines of cytoplasmic restorer lines of Brassica napus radish are particularly important for accelerating the breeding process. The present invention aims to provide a set of molecular markers closely related to the cytoplasmic restorer line 206R of Brassica napus radish for rapid breeding and identification of the cytoplasmic restorer line 206R of Brassica napus radish. Summary of the invention

[0005] The purpose of the present invention is to provide a set of molecular markers tightly linked to the restoration fragment of the radish cytoplasmic restoration line 206R material of Brassica napus, which can effectively distinguish other Brassica napus (such as R2000, Zhongshuang 11 material, and Bing 409), and can also identify the pure lines and heterozygous plants of 206R for use in Brassica napus breeding selection.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The molecular markers used for identifying the Brassica napus radish cytoplasmic restorer line 206R include two molecular markers SNP1 and SNP2. The nucleotide sequence of the SNP1 molecular marker is shown in SEQ ID NOs.7 and 8, and the polymorphic site is T or C. The nucleotide sequence of the SNP2 molecular marker is shown in SEQ ID NOs.9 and 10, and the polymorphic site is C or G. The pure and heterozygous states of the 206R restorer line can be effectively identified.

[0008] The amplification primer sequences of the SNP1 molecular marker are shown in SEQ ID NOs. 1-3, and the amplification primer sequences of the SNP2 molecular marker are shown in SEQ ID NOs. 4-6.

[0009] The application of the above molecular markers or primers in the breeding and identification of the Brassica napus radish cytoplasmic restorer line 206R and its hybrids: the genotype of the Brassica napus radish cytoplasmic restorer line 206R pure line at the polymorphic site of the SNP1 molecular marker is T:T, and the genotype of the polymorphic site of the SNP2 molecular marker is C:C; the genotype of the Brassica napus radish cytoplasmic restorer line 206R hybrid at the polymorphic site of the SNP1 molecular marker is T:C, and the genotype of the polymorphic site of the SNP2 molecular marker is C:G. Specifically, the following steps are included:

[0010] (1) extracting DNA from Brassica napus to be tested;

[0011] (2) using the above DNA as a template and a primer combination to perform a KASP reaction;

[0012] (3) The sample genotype is determined according to the fluorescence signal of the KASP product. If the genotype of the polymorphic site of the SNP1 molecular marker is T:T and the genotype of the polymorphic site of the SNP2 molecular marker is C:C, then it is the homozygous Brassica napus radish cytoplasmic restorer line 206R; if the genotype of the polymorphic site of the SNP1 molecular marker is T:C and the genotype of the polymorphic site of the SNP2 molecular marker is C:G, then it is the heterozygous Brassica napus radish cytoplasmic restorer line 206R; if the genotype of the polymorphic site of the SNP1 molecular marker is C:C and the genotype of the polymorphic site of the SNP2 molecular marker is G:G, then it is not the Brassica napus radish cytoplasmic restorer line 206R.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] (1) Strong specificity: The developed SNP1 and SNP2 dual marker system is closely linked to the 206R restorer genome recovery fragment, and can accurately distinguish its homozygous (T:T / C:C) and heterozygous (T:C / C:G) genotypes. It has the ability to specifically exclude other Brassica napus varieties (genotype C:C / G:G) such as R2000, Zhongshuang 11, and Bing 409.

[0015] (2) High accuracy: Compared with traditional SSR or AFLP markers, the fluorescence detection system based on KASP technology has a detection sensitivity of more than 99%, which can accurately identify single nucleotide polymorphism differences and avoid false positive interference.

[0016] (3) Efficient operation: Only two pairs of primers are needed to complete the identification. Compared with the traditional method that requires the use of multiple markers (such as 15-20 SSR marker screening), the detection time is shortened to 4 hours, and 384 samples can be processed at a time, greatly improving the breeding screening efficiency.

[0017] (4) Outstanding practicality in breeding: It can quickly identify homozygous restorer plants (for seed preservation) and heterozygous plants (for hybrid seed production) at the seedling stage, shortening the backcross breeding cycle by 2-3 generations; through the co-dominant marker characteristics of SNP1 (T / C) and SNP2 (C / G), the restorer genotype and background selection are simultaneously optimized, increasing the purity of the restorer line from 85% of the traditional method to 99.5%.

[0018] (5) Strong technical compatibility: The marker detection system is suitable for conventional PCR instruments and mainstream fluorescence detection platforms (such as LGC Genomics). It does not require special equipment and can be standardized across laboratories, providing reliable technical support for commercial seed production.

[0019] (6) Innovation: For the first time, a double SNP marker combination was developed for the cytoplasmic male sterility restorer line 206R of Brassica napus radish, filling the technical gap of the lack of specific molecular markers in this restorer line and solving the industry pain points of delayed phenotypic identification (flowering period observation is required) and inefficient background selection in traditional backcross breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the typing result of KASP marker primer 206RKASP1 in Example 2.

[0021] Figure 2 This is the typing result of KASP marker primer 206RKASP1 in Example 2. DETAILED DESCRIPTION

[0022] Example 1: Development of specific molecular markers and primers for the cytoplasmic restorer line 206R of Brassica napus radish

[0023] 1. Obtaining specific SNPs linked to the 206R restoration gene

[0024] 1. Sequencing data acquisition: The resequencing data of ZS11 and R2000 of Brassica napus and the resequencing data of the mixed pool of fertile and sterile plants in the 206R near-isogenic line population were used, and the BWA software was used to align them to the ZS11 genome (version V1, http: / / cbi.hzau.edu.cn / bnapus / index.php). SNPs were extracted and filtered by Samtools and VariScan2 software to obtain reliable SNP difference sites. The △SNP-Index method was used to search for the difference intervals in the mixed pool of fertile and sterile plants. Conventional Brassica napus such as Bing 409 and ZS11 were used as controls, and the sequencing data of 206R were compared with IGV software. Specific SNPs were screened in the flanking sequences of the restoration genes. The screening criteria were that the genotypes of conventional Brassica napus such as Bing 409 and ZS11 were consistent, and the genotypes of conventional Brassica napus such as Bing 409 and ZS11 were polymorphic with 206R. According to the above criteria, a total of 2 differential SNP markers were obtained, as shown in Table 1.

[0025] Table 1 Statistics of 2 differential SNP markers

[0026]

[0027] 2. DNA extraction, as follows: ① Take about 2 sections of 1.5 cm long leaves or seeds and put them into a 1.3 mL 96-well plate, and place the 96-well plate in a -80°C refrigerator for 4 hours or more; ② Place the 96-well plate in an oven and dry it at 65°C for 10 hours or more. After drying, take out the 96-well plate and add 2 4 mm steel balls to each well of the 96-well plate; ③ Quickly seal it with a silica gel film, place it in a tissue grinder, adjust the speed to 1500 rpm and grind for 1 minute. The grinding time can be appropriately extended. After grinding, place it in a deep well plate centrifuge for a split second, and centrifuge the ground tissue to ④ Take out the 96-well plate, carefully tear open the silica gel plate, add 500 μL of Tris-HCl extract to each well, then cover with a new silica gel plate and place the 96-well plate on a vortex oscillator for proper oscillation; ⑤ Place the 96-well plate in an oven set to 75°C for a warm bath for about 40 minutes; ⑥ After the warm bath, take out the 96-well plate, place it in a deep-well plate refrigerated centrifuge, adjust the speed to 4000 rpm and the temperature to room temperature, and centrifuge for 10 minutes; ⑦ After the centrifugation, take out the 96-well plate, extract 190 μL of the supernatant to another 0.8 mL deep-well plate refrigerated centrifuge pre-added with 190 μL of isopropanol 96-well plate, and seal it with a silicone film, oscillate it on a vortex oscillator for several times, and then place it in a -20℃ refrigerator to precipitate for 1 hour or more;⑧ Take out the 96-well plate and place it in a deep-well plate refrigerated centrifuge, adjust the speed to 4000rpm and the temperature to 4℃ and centrifuge for 10 minutes;⑨ Remove the supernatant and place the 96-well plate in a 65℃ oven for 30 minutes to dry it;⑩ Take out the 96-well plate, add 500μL ultrapure water to each well, cover it with a silicone cover, oscillate it on a vortex oscillator for several times, and then place it at room temperature overnight. It can be used overnight.

[0028] 2. KASP marker primer development

[0029] KASP marker primers were developed based on the two screened SNP markers, and then FAM and HEX fluorescent linker sequences were added to the 5' ends of the two forward primers, respectively. The primers developed for SNP1 were named 206RKASP1, including two forward primers and a universal reverse primer, and the primer sequences are shown in SEQ ID NOs. 1-3. The primers developed for SNP2 were named 206RKASP2, including two forward primers and a universal reverse primer, and the primer sequences are shown in SEQ ID NOs. 4-6.

[0030] Example 2: Molecular identification of the cytoplasmic restorer line 206R of Brassica napus

[0031] 116 conventional Brassica napus, 21 Brassica napus radish cytoplasmic restorer lines 206R, and 10 hybrids (206R and 10 maintainer lines) were selected for molecular identification using the KASP marker primers developed in Example 1. The reaction system is shown in Table 2.

[0032] Table 2 KASP reaction system

[0033]

[0034] Touchdown PCR reaction conditions are:

[0035] Pre-denaturation at 94°C for 15 min; denaturation at 95°C for 20 s, gradient annealing 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.

[0036] KASP genotyping results Figure 1 and Figure 2 As shown, Figure 1 Middle red dots represent homozygous genotype T:T, blue dots represent homozygous genotype C:C, and purple dots represent heterozygous genotype T:C; Figure 2 The red dots represent the homozygous genotype C:C, the blue dots represent the homozygous genotype G:G, and the purple dots represent the heterozygous genotype C:G. The genotype T:T of the SNP1 site indicates a pure line with the 206R restoration gene, the genotype C:C indicates that the R206 restoration gene is not present, and the genotype T:C indicates that the 206R restoration gene is heterozygous; the genotype C:C of the SNP2 site indicates a pure line with the R206 restoration gene, the genotype G:G indicates that the R206 restoration gene is not present, and the genotype C:G indicates that the R206 restoration gene is heterozygous. The two SNP sites specifically co-segregate with the restoration gene of the Brassica napus radish cytoplasmic restoration line R206. The genotypes of the two sites can accurately identify the Brassica napus radish cytoplasmic restoration line R206 and its hybrids.

[0037] Table 3 Genotyping results of markers R206KASP1 and R206KASP2

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] As shown in Table 3, the genotypes of 21 Brassica napus radish cytoplasmic restorer lines 206R at SNP1 were T:T, and the genotypes of SNP2 were C:C, the genotypes of 10 hybrids at SNP1 were T:C, and the genotypes of SNP2 were C:G, and the genotypes of 116 conventional Brassica napus cytoplasmic restorer lines 206R and their hybrids were C:C at SNP1 and G:G at SNP2. Therefore, the Brassica napus radish cytoplasmic restorer lines 206R and their hybrids can be accurately distinguished from other varieties using SNP1 and SNP2 loci.

[0044] 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 molecular marker for identifying the cytoplasmic restorer line 206R of Brassica napus radish, characterized in that: It includes two molecular markers, SNP1 and SNP2. The nucleotide sequence of the SNP1 molecular marker is shown in SEQ ID NOs. 7 and 8, and the polymorphic site is T or C. The nucleotide sequence of the SNP2 molecular marker is shown in SEQ ID NOs. 9 and 10, and the polymorphic site is C or G.

2. Primers for amplifying the molecular marker according to claim 1.

3. The primer according to claim 2, characterized in that The amplification primer sequences of the SNP1 molecular marker are shown in SEQ ID NOs. 1-3, and the amplification primer sequences of the SNP2 molecular marker are shown in SEQ ID NOs. 4-6.

4. Use of the molecular marker described in claim 1 in the breeding and identification of the Brassica napus radish cytoplasmic restorer line 206R and its hybrids.

5. Use of the primers described in claim 2 in the breeding and identification of the Brassica napus radish cytoplasmic restorer line 206R and its hybrids.

6. The use according to claim 4 or 5, characterized in that: The genotype of the Brassica napus radish cytoplasmic restorer line 206R pure line at the polymorphic site of the SNP1 molecular marker is T:T, and the genotype of the polymorphic site of the SNP2 molecular marker is C:C; the genotype of the Brassica napus radish cytoplasmic restorer line 206R hybrid at the polymorphic site of the SNP1 molecular marker is T:C, and the genotype of the polymorphic site of the SNP2 molecular marker is C:G.

7. The use according to claim 6, characterized in that: The following steps are involved: (1) Extracting DNA from the Brassica napus strain to be tested; (2) using the above DNA as a template and using the primer combination described in claim 3 to perform a KASP reaction; (3) The sample genotype is determined based on the fluorescence signal of the KASP product. If the genotype of the polymorphic site of the SNP1 molecular marker is T:T and the genotype of the polymorphic site of the SNP2 molecular marker is C:C, then it is the homozygous Brassica napus radish cytoplasmic restorer line 206R; if the genotype of the polymorphic site of the SNP1 molecular marker is T:C and the genotype of the polymorphic site of the SNP2 molecular marker is C:G, then it is the heterozygous Brassica napus radish cytoplasmic restorer line 206R; if the genotype of the polymorphic site of the SNP1 molecular marker is C:C and the genotype of the polymorphic site of the SNP2 molecular marker is G:G, then it is not the Brassica napus radish cytoplasmic restorer line 206R.

Images