A SNP molecular marker related to salt tolerance of rapeseed and its application

Abstract

The present invention belongs to the technical field of molecular genetic breeding, and specifically relates to a SNP molecular marker related to the salt tolerance of rapeseed and its application. The present invention provides a SNP molecular marker related to the salt tolerance of rapeseed, the SNP molecular marker corresponds to the 4347264bp of the fifth chromosome of the Zhongshuang 11 (ZS11) Brassica napus genome, and the base is A / C; the SNP molecular marker provided by the present invention has potential application value in the molecular breeding of Brassica napus salt stress response, and the SNP molecular marker provided by the present invention can be used to quickly screen salt-tolerant rapeseed materials, reduce breeding costs, save labor costs, and greatly improve selection efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular genetic breeding, and in particular relates to a SNP molecular marker related to the salt tolerance of rapeseed and an application thereof. Background Art

[0002] Soil salinization, as one of the key abiotic stresses in global crop production, seriously threatens agricultural production and food security. With global warming and unreasonable farming practices, it is estimated that by 2050, more than 50% of cultivated land will face salt stress, especially in arid and semi-arid areas. my country has nearly 1.5 billion mu of saline-alkali land resources, of which about 299 million mu can be developed and utilized. These saline-alkali lands include saline-alkali barrier cultivated land that has not been managed and transformed, as well as unused and newly formed saline-alkali wasteland, which are huge in scale. Reasonable transformation and utilization of these lands will greatly enhance agricultural production capacity. It is estimated that the output of grain, cotton and oil can be increased by more than 20 billion jin each year, which has huge development and management potential.

[0003] At present, my country has developed technologies in fields including water conservancy engineering, biology, agronomy and chemistry to manage and utilize saline-alkali land. Among them, the cultivation of salt-tolerant crops through biotechnology is considered to be one of the most efficient and environmentally friendly methods. However, since the salt tolerance of plants is affected by a variety of genetic factors, there are significant differences in salt tolerance between different plant species and varieties, which involve complex signal transduction and molecular regulatory networks. As an important oil crop, rapeseed is significantly more salt-tolerant than rice and wheat, and has a wide range of adaptability and rich stress resistance resources. The completion of whole genome sequencing has made it one of the ideal model crops for studying the mechanism of crop salt tolerance and genetic improvement. In the situation where the total cultivated land area continues to tighten and the task of maintaining food security is arduous, how to give full play to the characteristics of rapeseed, tap saline-alkali land resources without competing with food, and improve my country's self-sufficiency rate of oil and fat is currently a technical problem that needs to be solved urgently.

[0004] Studies in recent years have shown that molecular markers and candidate genes related to plant abiotic stress can be discovered through genome-wide association analysis (GWAS), which provides an important reference for revealing the molecular regulatory mechanism of crops to salt stress. GWAS technology has become a powerful tool for studying the relationship between complex quantitative traits and physiological phenotypes. It can help identify the salt tolerance of rapeseed germplasm and provide technical support for molecular marker-assisted breeding and precision breeding of salt-tolerant crops. Therefore, the development of reliable molecular markers through technical methods such as GWAS not only has important theoretical significance, but also has great practical application value. Summary of the invention

[0005] The purpose of the present invention is to develop a molecular marker that is closely linked to the salt tolerance of rapeseed, so as to achieve efficient and rapid screening of salt-tolerant rapeseed germplasm.

[0006] The invention provides a SNP molecular marker related to the salt tolerance of rapeseed, wherein the SNP molecular marker corresponds to 4347264 bp of chromosome 5 of Zhongshuang 11 cabbage rapeseed genome, and the base is A / C.

[0007] The present invention also provides a primer set for amplifying the SNP molecular marker described in the above technical solution, the primer set comprising an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2.

[0008] The present invention also provides the use of the SNP molecular marker described in the above technical solution or the primer set described in the above technical solution in screening salt-tolerant rapeseed germplasm.

[0009] The present invention also provides the use of the SNP molecular marker described in the above technical solution or the primer set described in the above technical solution in assisting the creation of salt-tolerant rapeseed.

[0010] Preferably, the rapeseed includes Brassica napus.

[0011] The present invention also provides a kit for screening salt-tolerant rapeseed germplasm, the kit comprising the primer set described in the above technical solution.

[0012] Preferably, the kit further comprises a positive standard; the nucleotide sequence of the positive standard is shown as SEQ ID NO.3.

[0013] The present invention also provides a method for screening salt-tolerant rapeseed germplasm, comprising the following steps:

[0014] The genomic DNA of the rapeseed to be tested is amplified by PCR using the primer set described in the above technical solution. If the size of the PCR amplification product is 2043 bp, the rapeseed to be tested is a material with strong salt tolerance; if there is no band at the 2043 bp position of the PCR product, the rapeseed to be tested is a material with weak salt tolerance.

[0015] Preferably, the reaction system for PCR amplification is: 1 μL of 50 ng / μL DNA template, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 10 μL of 2×PCRMasterMix, and 7 μL of ddH2O.

[0016] Preferably, the PCR amplification program is: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 10 min.

[0017] Beneficial Effects

[0018] The present invention provides a SNP molecular marker related to the salt tolerance of rapeseed, and the SNP molecular marker corresponds to the 4347264bp of the fifth chromosome of the Zhongshuang 11 cabbage rapeseed genome, and the base is A / C. As can be seen from the embodiment, the present invention obtains the main effect loci of the salt tolerance trait of rapeseed by conducting phenotypic investigation and statistics on the salt tolerance of seedlings of 350 rapeseed resource groups, and combines the resequencing genotype data for GWAS analysis, and develops molecular markers related to the salt tolerance of rapeseed; the SNP molecular marker provided by the present invention has potential application value in the molecular breeding of cabbage rapeseed salt stress response, can quickly screen salt-tolerant rapeseed materials, reduce breeding costs, save labor costs, and greatly improve selection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 The distribution map of salt tolerance index of 346 rapeseed resource populations;

[0021] Figure 2 Manhattan plot of GWAS results of salt tolerance of 346 rapeseed resource populations;

[0022] Figure 3 The agarose gel electrophoresis image shows the PCR results of molecular markers in 48 rapeseed materials;

[0023] Figure 4 A comparison chart of the salt tolerance of different haplotypes of molecular markers. DETAILED DESCRIPTION

[0024] The present invention provides a SNP molecular marker related to the salt tolerance of rapeseed, and the SNP molecular marker corresponds to the 4347264bp of the fifth chromosome of the Zhongshuang 11 cabbage rape genome, and the base is A / C. In the present invention, when the SNP molecular marker is A, it indicates that the salt tolerance of the rapeseed material is relatively high; when the SNP molecular marker is C, it indicates that the salt tolerance of the rapeseed material is relatively low. The present invention preferably develops a DNA fragment based on the SNP molecular marker, which is related to the salt tolerance of rapeseed. The nucleotide sequence of the DNA fragment of the present invention is shown in SEQ ID NO.3. The judgment standard of the salt tolerance of the present invention is preferably the fresh weight ratio of rapeseed seedlings under 150mMNaCl hydroponic salt stress conditions and rapeseed seedlings under normal growth conditions (fresh weight ratio=fresh weight of salt-treated plants / fresh weight of control group plants×100%).

[0025] The present invention also provides a primer set for amplifying the SNP molecular marker described in the above technical solution, the primer set comprising an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, specifically: 5'-ATTATGGTTTGGATTTTGTAATTA-3'; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2, specifically: 5'-TCAAGGCATAGGACATACTAT TTTTGTC-3'.

[0026] The present invention also provides the use of the SNP molecular marker described in the above technical solution or the primer set described in the above technical solution in screening salt-tolerant rapeseed germplasm.

[0027] The present invention also provides the use of the SNP molecular marker described in the above technical solution or the primer set described in the above technical solution in assisting the creation of salt-tolerant rapeseed.

[0028] In the present invention, the rapeseed preferably includes Brassica napus.

[0029]

[0030] The present invention also provides a method for screening salt-tolerant rapeseed germplasm, comprising the following steps:

[0031] The genomic DNA of the rapeseed to be tested is amplified by PCR using the primer set described in the above technical solution. If the size of the PCR amplification product is 2043 bp, the rapeseed plant to be tested is a material with strong salt tolerance; if there is no band at the 2043 bp position of the PCR product, the rapeseed plant to be tested is a material with weak salt tolerance.

[0032] In the present invention, the reaction system of the PCR amplification is preferably: 1 μL of 50 ng / μL DNA template, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 10 μL of 2×PCRMasterMix, and 7 μL of ddH2O; the program of the PCR amplification is preferably: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 10 min.

[0033] After the present invention uses the above primer set to perform PCR amplification on different rapeseed genotypes, it is preferred to detect the presence or absence of the target PCR product band by agarose gel electrophoresis. The method provided by the present invention can effectively screen rapeseed planting resources with different salt tolerance, specifically calculating the fresh weight ratio of salt-treated rapeseed seedlings and rapeseed seedlings in normal growth state under hydroponic conditions. The fresh weight ratio is higher than 54.20% for a high fresh weight ratio, and the fresh weight ratio is lower than 54.20% for a low fresh weight ratio; the rapeseed material with a band at the 2043bp position of the PCR product has a higher salt tolerance and is a rapeseed material with a high fresh weight ratio; and the rapeseed material with no band at the 2043bp position of the PCR product has a lower salt tolerance and is a rapeseed material with a low fresh weight ratio. The present invention does not specifically limit the method for obtaining the rapeseed genomic DNA to be tested, and the conventional extraction method in the industry can be used. In the embodiment of the present invention, the CTAB method is preferably used to extract the rapeseed genomic DNA.

[0034] To further illustrate the present invention, a SNP molecular marker related to the salt tolerance of rapeseed and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] Detection of salt tolerance index of rapeseed germplasm population

[0037] A total of 346 rapeseed germplasm resource groups with different phenotypes and genetic stability were used as samples. The rapeseed seeds were treated with 84 disinfectant for 10 minutes and then washed three times with sterile water.

[0038] The sterilized seeds were placed in a 4°C refrigerator overnight, and then evenly sown in a petri dish covered with three layers of filter paper for germination. After 3 days, the uniformly germinated seedlings were divided into a control group and a salt treatment group, and transplanted into black hydroponic boxes. The hydroponic nutrient solution was 1 / 2 Hoagland's nutrient solution, and the hydroponic nutrient solution was changed every two days. The growth environment was a standard plant growth room, RH60%, 24 / 22°C, light-dark ratio 16 / 8h, 120μmol·m -2 ·s -1 .

[0039] After 8 days of growth, the original hydroponic nutrient solution was replaced for the plants in the control group, and the solution was replaced every two days during the culture period; the nutrient solution of the plants in the salt treatment group was replaced with a hydroponic nutrient solution containing 150 mM NaCl, and the salt treatment was performed, and the hydroponic nutrient solution containing 150 mM NaCl was replaced every two days during the culture period;

[0040] After growing for another 8 days, take the whole rapeseed plant, use absorbent paper to absorb the water at the root, weigh the individual plant, and calculate the fresh weight ratio according to the following formula:

[0041] Fresh weight ratio (salt tolerance index) = fresh weight of salt-treated plants / fresh weight of control group plants × 100%.

[0042] The distribution diagram of salt tolerance index of 346 rapeseed germplasm resource populations is as follows: Figure 1 shown.

[0043] Figure 1 The results of data analysis on the salt tolerance index of rapeseed germplasm resources in China showed that the distribution of rapeseed salt tolerance index was continuous, the variation distribution was normal, and the variation range was very wide, which proved that rapeseed salt tolerance was a quantitative trait. The median of the salt tolerance index in the rapeseed germplasm resources population was 54.20%, and the coefficient of variation was 29.72%. Therefore, the salt tolerance index of rapeseed germplasm in this population was significantly higher than 54.20%, which was defined as high salt tolerance, and lower than 54.20%, which was defined as low salt tolerance.

[0044] Example 2

[0045] Extraction of DNA from Leaves of Rapeseed Resource Population

[0046] The total DNA of the rapeseed leaves in Example 1 was extracted using the CTAB method. After the rapeseed DNA was extracted, the rapeseed resource population was resequenced, and a GWAS analysis of the salt tolerance index was performed.

[0047] SNP variation analysis was performed on the genotype data of rapeseed resource populations. SNPs were detected using software such as AMTOOLS, and then the detected SNPs were annotated using ANNOVAR software. After obtaining the SNP variation, the mixed linear model (MLM) of Emmax software was used to perform GWAS analysis on the genotype data and phenotype data. The analysis results are shown in the figure. Figure 2 As shown. Salt tolerance refers to fresh weight ratio, molecular marker identification refers to electrophoresis bands, and the consistency of the two reflects the accuracy of the marker; the analysis results show that a SNP marker scaffoldA05_4347264 located on chromosome A05 is significantly associated with the relative fresh weight of rapeseed under salt stress. The SNP is located at the 4347264th base of chromosome A05 and can be repeatedly detected in three replicates. The SNP site is the difference in base A / C.

[0048] Example 3

[0049] Development of specific molecular markers linked to high salt tolerance traits in rapeseed

[0050] For the variable site scaffoldA05_4347264 obtained in Example 2, an upstream primer F and a downstream primer R were designed; the nucleotide sequence of the upstream primer F is shown in SEQ ID NO.1, specifically: 5'-ATTATGGTTTGGATTTTGTAATTA-3'; the nucleotide sequence of the downstream primer R is shown in SEQ ID NO.2, specifically: 5'-TCAAGGCATAGGACATACTATTTTTGTC-3'. The target fragment amplified using the primer pair is shown in SEQ ID NO.3.

[0051] Example 4

[0052] Application of molecular markers in breeding of rapeseed with high salt tolerance

[0053] 48 materials (including rape varieties such as Huzhe Shuang 8, Hua Shuang 4, Su You 3, You Yan 2, Hua You 13, and Su You 5) were randomly selected from the 346 rape core germplasms in Example 1, and genomic DNA was extracted from the leaves of rape seedlings. The genomic DNA was used as a template and PCR amplification was performed using the specific molecular marker primer pair combination (upstream primer F and downstream primer R) designed in Example 3. The PCR reaction system was (20 μL system): 1 μL DNA template (concentration of 50 ng / μL), 1 μL of upstream and downstream primers (both concentrations of 10 μM), 10 μL of 2×PCR Master Mix, and 7 μL of ddH2O. The PCR amplification program was as follows: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 10 min. The PCR product was detected by electrophoresis using 1 wt.% agarose gel. The material that can detect the target band (2043bp) has a higher salt tolerance, while the material that cannot detect the target band (2043bp) has a lower salt tolerance. Figure 3 And as shown in Table 1.

[0054] Table 1 Salt tolerance index of rapeseed germplasm

[0055]

[0056]

[0057] Depend on Figure 3 It can be seen that when the molecular markers provided by the present invention were used to screen the salt tolerance of rapeseed, PCR bands were detected in samples No. 1, 3, 5, 6, 8, 10, 11, 12, 13, 14, 18, 20, 21, 22, 24, 25, 27, 29, 31, 34, 35, 36, 38, 40, 41, 43, 45, 47, and 48, and no PCR bands were detected in samples No. 2, 4, 7, 9, 15, 16, 17, 19, 23, 26, 28, 30, 32, 33, 37, 39, 42, 44, and 46. The relative fresh weight statistical analysis is shown in Table 1. The average salt tolerance index of the germplasm number with higher salt tolerance obtained by molecular marker-assisted selection is 74.83%; the average salt tolerance index of the germplasm number with lower salt tolerance obtained by molecular marker-assisted selection is 37.55%, and the two have reached a significant difference level (p value = 1.6374E-11). The correlation coefficient between the presence or absence of the band and the level of salt tolerance is 0.79, indicating that the salt-tolerant molecular marker has a good screening effect ( Figure 4 ).

[0058] In summary, the use of the SNP molecular markers closely associated with the salt tolerance of rapeseed in the present invention can realize the screening of high salt-tolerant germplasm in the rapeseed resource population, predict the salt tolerance of rapeseed, and improve the efficiency of salt-tolerant rapeseed breeding.

[0059] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer set for amplifying SNP molecular markers related to salt tolerance in rapeseed, characterized in that: The primer set includes an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2; the rapeseed salt tolerance-related SNP molecular marker corresponds to the 23rd position of the nucleotide sequence shown in SEQ ID NO.3, and the base is A / C.

2. Application of a reagent for detecting SNP molecular markers associated with salt tolerance of rapeseed in screening salt-tolerant rapeseed germplasm; the SNP molecular marker associated with salt tolerance of rapeseed corresponds to the 23rd position of the nucleotide sequence shown in SEQ ID NO.3, and the base is A / C; when the SNP molecular marker is A, the rapeseed material has strong salt tolerance; when the SNP molecular marker is C, the rapeseed material has weak salt tolerance.

3. Application of a reagent for detecting SNP molecular markers associated with salt tolerance of rapeseed in assisting the creation of salt-tolerant rapeseed; the SNP molecular marker associated with salt tolerance of rapeseed corresponds to the 23rd position of the nucleotide sequence shown in SEQ ID NO.3, and the base is A / C; when the SNP molecular marker is A, the rapeseed material has strong salt tolerance; when the SNP molecular marker is C, the rapeseed material has weak salt tolerance.

4. The use according to claim 2 or 3, characterized in that: The reagents include the primer set according to claim 1.

5. The use according to claim 2 or 3, characterized in that: The rapeseed includes Brassica napus.

6. A kit for screening salt-tolerant rapeseed germplasm, characterized in that: The kit comprises the primer set according to claim 1.

7. The kit according to claim 6, characterized in that The kit also includes a positive standard; the nucleotide sequence of the positive standard is shown in SEQ ID NO.

3.

8. A method for screening salt-tolerant rapeseed germplasm, characterized in that: The following steps are involved: The genomic DNA of the rapeseed to be tested is amplified by PCR using the primer set described in claim 1, and the genotype is determined based on the amplified product. If the genotype of the SNP molecular marker related to the salt tolerance of the rapeseed is A, the rapeseed to be tested is a material with strong salt tolerance; if the genotype of the SNP molecular marker related to the salt tolerance of the rapeseed is C, the rapeseed to be tested is a material with weak salt tolerance.

9. The method according to claim 8, characterized in that The reaction system of the PCR amplification is: 1 μL of 50 ng / μL DNA template, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 10 μL of 2×PCR MasterMix, and 7 μL of ddH2O.

10. The method according to claim 8 or 9, characterized in that: The PCR amplification program is: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 10 min.

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