A set of molecular markers for specific detection of Brassica napus cytoplasmic male restorer line 206R and its application

Abstract

The application discloses a set of molecular markers for specifically detecting a Brassica napus radish cytoplasmic restorer line 206R and application of the molecular markers, comprising two SNP molecular markers, and the SNP sites are located at the 26276606th position and the 26190259th position of an A10 chromosome. A double-SNP marker combination is first developed for the Brassica napus radish cytoplasmic male sterility restorer line 206R, a technical blank of the restorer line lacking specific molecular markers is filled, and the double-SNP marker combination can be used for distinguishing the presence or absence of the restorer gene site and the pure or hybrid state of a single plant in offspring of the restorer line, thereby assisting breeding selection.

Description

Technical Field

[0001] The invention belongs to the technical field of rapeseed breeding and molecular identification, and particularly relates to a set of molecular markers for specifically detecting a cytoplasmic restorer line 206R of Brassica napus radish and applications thereof. Background Art

[0002] Brassica napus has significant hybrid vigor. Compared with conventional varieties, the use of rapeseed hybrids can increase yield, enhance adaptability, and improve stress resistance. During the hybrid breeding process, the purity of the hybrids will directly affect the quality and quality of the variety. At present, the main methods for producing rapeseed hybrids in my country include 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 most effective way to utilize hybrid vigor in rapeseed. The types of cytoplasmic male sterility that have been discovered or created so far mainly include Polima cytoplasmic male sterility, Shaanxi 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 Institute of Agricultural Sciences (INRA) created the radish cytoplasmic restorer line R2000 through radiation induction, leading to its widespread use in Europe. However, due to limitations in the patent for this restorer line, its early use in rapeseed breeding in China was limited. In previous studies, through distant hybridization combined with backcrossing, we successfully bred a new radish cytoplasmic restorer line, 206R (CN113016606A), for rapeseed. Compared to the French R2000, this restorer line has a shorter radish chromosome segment and is located on the A10 linkage group of rapeseed. It has excellent intergenerational stability, sufficient pollen, a good recovery rate, and normal seed set, among other advantages, making it directly applicable to rapeseed hybrid vigor breeding research in my country.

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

[0005] The purpose of the present invention is to provide a set of molecular markers that are tightly linked to the restoration fragment of the radish cytoplasmic restorer line 206R material of Brassica napus, which can effectively distinguish it from other Brassica napus materials (such as R2000, Zhongshuang 11 material, and Bing 409), and can also identify 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. These markers can effectively identify the pure and heterozygous states of the 206R restorer line.

[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 Brassica napus radish cytoplasmic restorer line 206R pure line has a genotype of T:T at the polymorphic site of the SNP1 molecular marker and a genotype of C:C at the polymorphic site of the SNP2 molecular marker; the Brassica napus radish cytoplasmic restorer line 206R hybrid has a genotype of T:C at the polymorphic site of the SNP1 molecular marker and a genotype of C:G at the polymorphic site of the SNP2 molecular marker. The specific steps include:

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

[0011] (2) Using the above DNA as a template, a KASP reaction was performed using a primer combination;

[0012] (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.

[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 genomic restoration fragment of the 206R restorer line, and can accurately distinguish its homozygous (T:T / C:C) and heterozygous (T:C / C:G) genotypes. It has the specific ability to exclude other Brassica napus varieties (genotype C:C / G:G) such as R2000, Zhongshuang 11, and Bing 409.

[0015] (2) High accuracy: The fluorescence detection system based on KASP technology has a detection sensitivity of more than 99% compared to traditional SSR or AFLP markers, 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 single plants (for seed preservation) and heterozygous single plants (for hybrid seed production) in the restorer line 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, and the purity of the restorer line is increased from 85% of the traditional method to 99.5%.

[0018] (5) Strong technical compatibility: The labeling 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 (needing to observe during flowering) 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

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

[0024] 1. Sequencing Data Acquisition: Resequencing data from Brassica napus ZS11 and R2000, as well as resequencing data from a pool of fertile and sterile plants from the 206R near-isogenic line population, were mapped to the ZS11 genome (version 1, http: / / cbi.hzau.edu.cn / bnapus / index.php) using BWA software. SNPs were extracted and filtered using Samtools and VariScan2 software to identify reliable SNP differential sites. The ΔSNP-Index method was used to search for differential intervals within the pool of fertile and sterile plants. Sequencing data from conventional Brassica napus varieties, such as Bing 409 and ZS11, were used as controls and aligned with the 206R sequencing data using IGV software. Specific SNPs were screened for in the flanking sequences of the restorer gene. The screening criteria required that the genotypes of conventional Brassica napus varieties, such as Bing 409 and ZS11, were identical and polymorphic to those of 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 two differential SNP markers

[0026]

[0027] 2. DNA extraction, specifically as follows: ① Take about 2 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 with a silicone film, place 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 short time, 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 shaker for appropriate shaking; 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 and 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 and extract 190 μL of the supernatant into another 0.8 mL deep-well plate pre-added with 190 μL of isopropanol. Prepare a 96-well plate, seal it with a silicone film, vortex it several times, and then place it in a -20°C refrigerator to settle for 1 hour or more; ⑧ Remove the 96-well plate and place it in a deep-well plate refrigerated centrifuge, adjust the speed to 4000 rpm and the temperature to 4°C, and centrifuge for 10 minutes; ⑨ Remove the supernatant and place the 96-well plate in a 65°C oven to dry it for 30 minutes; ⑩ Remove the 96-well plate, add 500 μL of ultrapure water to each well, cover it with a silicone lid, vortex it 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. FAM and HEX fluorescent linker sequences were added to the 5' ends of the two forward primers, respectively. The primers developed for SNP1, named 206RKASP1, include two forward primers and a universal reverse primer. The primer sequences are shown in SEQ ID NOs. 1-3. The primers developed for SNP2, named 206RKASP2, include two forward primers and a universal reverse primer. The primer sequences are shown in SEQ ID NOs. 4-6.

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

[0031] 116 conventional Brassica napus accessions, 21 Brassica napus accessions, 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 each cycle; denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, 30 cycles.

[0036] KASP genotyping results are as follows 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 Red dots represent the homozygous genotype C:C, blue dots represent the homozygous genotype G:G, and purple dots represent the heterozygous genotype C:G. Genotype T:T at SNP1 indicates a pure line containing the 206R restorer gene, genotype C:C indicates the absence of the R206 restorer gene, and genotype T:C indicates the 206R restorer gene is heterozygous. Genotype C:C at SNP2 indicates a pure line containing the R206 restorer gene, genotype G:G indicates the absence of the R206 restorer gene, and genotype C:G indicates the R206 restorer gene is heterozygous. Both SNPs specifically cosegregate with the restorer gene of the Brassica napus (Brassica napus) radish cytoplasmic restorer line R206. The genotypes at these two loci can be used to accurately identify the Brassica napus radish cytoplasmic restorer 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, 21 Brassica napus radish cytoplasmic restorer line 206R had a T:T genotype at SNP1 and a C:C genotype at SNP2; 10 hybrids had a T:C genotype at SNP1 and a C:G genotype at SNP2; and 116 conventional Brassica napus cultivars had a C:C genotype at SNP1 and a G:G genotype at SNP2. Therefore, the Brassica napus radish cytoplasmic restorer line 206R and its hybrids can be accurately distinguished from other varieties using SNP1 and SNP2.

[0044] 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. 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. The primer for amplifying the molecular marker according to claim 1, 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.

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

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

5. The use according to claim 3 or 4, 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.

6. The use according to claim 5, 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 the primers described in claim 2 to perform a KASP reaction; (3) The genotype of the sample was determined based on the fluorescence signal of the KASP product. If the genotype of the polymorphic site of the SNP1 molecular marker was T:T and the genotype of the polymorphic site of the SNP2 molecular marker was C:C, then the sample was a homozygous Brassica napus radish cytoplasmic restorer line 206R; if the genotype of the polymorphic site of the SNP1 molecular marker was T:C and the genotype of the polymorphic site of the SNP2 molecular marker was C:G, then the sample was a heterozygous Brassica napus radish cytoplasmic restorer line 206R; if the genotype of the polymorphic site of the SNP1 molecular marker was C:C and the genotype of the polymorphic site of the SNP2 molecular marker was G:G, then the sample was not a Brassica napus radish cytoplasmic restorer line 206R.

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