Mustard SNP (Single Nucleotide Polymorphism) molecular marker combination and application thereof

Abstract

The invention discloses a mustard SNP (Single Nucleotide Polymorphism) molecular marker combination and application thereof. The mustard SNP molecular marker combination is composed of 1694 SNP molecular markers, and the physical positions of the 1694 SNP molecular markers are determined based on sequence alignment of a mustard reference genome Bju.gene. According to the scheme, the mustard SNP molecular marker combination marker is a co-dominant marker, and has the characteristics of high quality, single copy, high polymorphism, high specificity, high sensitivity and high resolution; the marking is not influenced by environmental conditions, seeds or any type of plant tissues can be used, the detection result is accurate, and the repeatability and the stability are good; different detection laboratories and different data results can be compared and verified, and the data has universal comparability, can be accurately used for background detection of mustard breeding materials, and has wide application universality.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a mustard SNP molecular marker combination and application thereof. Background Art

[0002] Mustard is one of the important winter oil crops and cash crops. It can not only be eaten directly as a vegetable, but its roots, stems and leaves also have relevant value. Its excellent drought-resistant and heat-resistant varieties have been planted on a large scale in many places. The research on mustard in China has been going on for a long time. However, due to the traditional interspecific breeding, mustard has been degenerated and it is difficult to select high-quality mustard varieties through conventional breeding. Therefore, it is urgent to develop new mustard variety selection tools.

[0003] At present, mustard breeding is basically conventional breeding, that is, breeding is carried out by phenotypic selection. Conventional phenotypic selection breeding depends on the visual recognition of morphological characteristics and biological characteristics. The investigation and identification of phenotypes mainly rely on the investigation of breeding samples after they show phenotypes during the growth process. The selection of offspring here requires waiting until the material grows to maturity and investigating each individual plant. The work is relatively cumbersome, the breeding efficiency is low, the judgment criteria are often difficult to accurately quantify, and they are highly subjective. The detection sensitivity and resolution are low; they are easily affected by the environment and cultivation conditions, and the accuracy and stability are poor; they are time-consuming and time-effective; they require a lot of manpower and material resources, and the cost is high. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a mustard SNP molecular marker combination.

[0005] The present invention also provides a primer set and / or probe for detecting the above mustard SNP molecular marker combination.

[0006] The invention also provides a liquid phase chip.

[0007] The present invention also provides a kit.

[0008] The present invention also provides an application of the above-mentioned mustard SNP molecular marker combination, primer set and / or probe, liquid phase chip or kit.

[0009] The invention also provides a mustard breeding method.

[0010] According to one aspect of the present invention, a mustard SNP molecular marker combination is proposed, which consists of 1694 SNP molecular markers. The physical positions of the 1694 SNP molecular markers are determined based on sequence alignment of the mustard reference genome Bju.genome, and the site information is specifically shown in Table 1 below.

[0011] Table 1

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033] In a second aspect of the present invention, a primer set and / or probe for detecting the above-mentioned mustard SNP molecular marker combination is provided.

[0034] In a third aspect of the present invention, a liquid phase chip is provided, wherein the liquid phase chip comprises the above-mentioned primer set and / or probe for detecting the mustard SNP molecular marker combination.

[0035] In a fourth aspect of the present invention, a kit is provided, wherein the kit comprises the above-mentioned primer set and / or probe for detecting the mustard SNP molecular marker combination.

[0036] In a fifth aspect of the present invention, an application of at least one of the above-mentioned mustard SNP molecular marker combination, liquid phase chip and kit is proposed, and the application is application in identification of mustard germplasm resources.

[0037] In some embodiments of the present invention, the application is application in genetic diversity assessment of Brassica juncea.

[0038] In some embodiments of the present invention, the application is application in purity identification of mustard varieties.

[0039] In some embodiments of the present invention, the application is application in authenticity identification of mustard varieties.

[0040] In some embodiments of the present invention, the application is application in the construction of genetic map and QTL positioning of Brassica juncea.

[0041] In some embodiments of the present invention, the application is application in genome-wide association analysis of Brassica juncea.

[0042] In some embodiments of the present invention, the application is application in molecular marker-assisted selection breeding of Brassica juncea.

[0043] In some embodiments of the present invention, the application is application in targeted improvement of Brassica juncea.

[0044] In some embodiments of the present invention, the application is application in multi-gene aggregation breeding of Brassica juncea.

[0045] In some embodiments of the present invention, the application is application in whole genome selection of Brassica juncea.

[0046] In some embodiments of the present invention, the application is application in molecular design breeding of Brassica juncea.

[0047] In a sixth aspect of the present invention, a method for detecting a combination of SNP molecular markers in Brassica juncea is provided, comprising the following steps:

[0048] 1) extracting genomic DNA of the mustard to be tested;

[0049] 2) using the above primer set and / or probe to perform multiplex PCR amplification on the genomic DNA of the mustard to be tested to obtain an amplified product;

[0050] 3) The genotype of the amplified product is measured by a second generation sequencing method to obtain the genotype of the mustard sample to be tested.

[0051] In some embodiments of the present invention, the multiplex PCR amplification includes a first round of PCR amplification and a second round of PCR amplification.

[0052] In some embodiments of the present invention, the reaction system of the first round of PCR amplification includes: 1-3 μL DNA, 0.5-2 μL mixed marker combination solution, 20-30 μL amplification enzyme and buffer solution MIX, and water is added to make up to 50 μL.

[0053] In some embodiments of the present invention, the reaction system of the first round of PCR amplification includes: 2 μL DNA, 1 μL mixed marker combination solution, 25 μL amplification enzyme and buffer solution MIX, and water is added to make up to 50 μL.

[0054] In some embodiments of the present invention, the reaction procedure of the first round of PCR amplification is as follows: 93-95°C for 8-12 min; entering the cycling stage: 93-95°C for 0.5-2 min, 58-62°C for 2-4 min, 70-74°C for 0.5-2 min, 35-45 cycles of amplification; 70-74°C for 18-22 min.

[0055] In some embodiments of the present invention, the reaction procedure of the first round of PCR amplification is as follows: 94°C for 10 min; entering the cycling stage: 94°C for 1 min, 60°C for 3 min, 72°C for 1 min, 40 cycles of amplification; 72°C for 20 min.

[0056] In some embodiments of the present invention, the reaction system of the second round of PCR amplification includes: 1-3 μL of the product obtained from the first round of PCR amplification, 0.5-2 μL of sequencing adapter, 10-20 μL of amplification enzyme and buffer mixture MIX, and water is added to make up to 30 μL.

[0057] In some embodiments of the present invention, the reaction system of the second round of PCR amplification includes: 2 μL of the product obtained from the first round of PCR amplification, 1 μL of sequencing adapter, 15 μL of amplification enzyme and buffer mixture MIX, and water is added to make up to 30 μL.

[0058] In some embodiments of the present invention, the reaction procedure of the second round of PCR amplification is as follows: 93-95°C for 8-12 min; entering the cycling stage: 93-95°C for 0.5-2 min, 58-62°C for 2-4 min, 70-74°C for 0.5-2 min, 8-12 cycles of amplification; 70-74°C for 18-22 min.

[0059] In some embodiments of the present invention, the reaction procedure of the second round of PCR amplification is as follows: 94°C for 10 min; entering the cycling stage: 94°C for 1 min, 60°C for 3 min, 72°C for 1 min, 10 cycles of amplification; 72°C for 20 min.

[0060] In a seventh aspect of the present invention, a breeding method for mustard is proposed, comprising the following steps: using the above detection method to detect the DNA of the mustard to be detected, and selecting mustard for subsequent breeding.

[0061] The present invention has at least the following beneficial effects: (1) The scheme of the present invention provides a 1.6K whole genome marker combination, which is a co-dominant marker with the characteristics of high quality, single copy, high polymorphism (the MAF values ​​in 671 mustard varieties are all higher than>=0.35), high specificity, sensitivity and resolution; the marker is not affected by environmental conditions, can be used with seeds or any type of plant tissue, and the detection results are accurate, repeatable and stable; different testing laboratories and different data results can be compared and verified with each other, the data has universal comparability, can be accurately used for background detection of mustard breeding materials, and has wide application universality.

[0062] (2) The SNP marker detection of the present invention is based on the combination of multiplex PCR + second-generation sequencing technology, which has great flexibility in detecting samples, low cost, and a short cycle, and can be popularized in seed production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0064] Figure 1 This is a flow chart of screening of the mustard SNP molecular marker combination in Example 1 of the present invention;

[0065] Figure 2 The distribution diagram of the mustard SNP molecular marker combination on chromosomes in Example 1 of the present invention;

[0066] Figure 3 This is a detection flow chart in Example 2 of the present invention;

[0067] Figure 4 This is a detection principle diagram in Example 2 of the present invention;

[0068] Figure 5 This is a cluster analysis diagram in Example 4 of the present invention. DETAILED DESCRIPTION

[0069] The following will be clearly and completely described in conjunction with the embodiments of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without paying creative work all belong to the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conditions recommended by the normal conditions or the manufacturers. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be obtained by commercial purchase.

[0070] Example 1

[0071] This embodiment provides a mustard SNP molecular marker combination.

[0072] 1. The steps for obtaining the mustard SNP molecular marker combination (the mustard 1.6K SNP molecular marker combination) are as follows: Figure 1 As shown, the details are as follows:

[0073] (1) Acquisition of mustard germplasm resource data

[0074] The mustard germplasm resource database was constructed using resequencing data extracted from 671 core mustard germplasm materials from Hunan Agricultural University.

[0075] (2) Extraction and screening of SNP loci in Brassica juncea

[0076] The data of 671 core germplasm resources were used for SNP extraction and polymorphism analysis, and finally 266,661 SNP sites were obtained. The 266,661 SNP sites collected were screened and compared with the reference mustard genome Bju.genome, and 1,694 good matching sites were obtained, and marker design was performed.

[0077] (3) Selection of 1694 whole genome marker combinations of Brassica juncea

[0078] The 1694 designed markers were further aligned with the reference genome of mustard (Bju. genome), and the marker sequence with the best specificity was selected for synthesis. The following figure shows the distribution of the sites on the chromosome of mustard. The information of the 1694 sites and the specific base sites are shown in Table 1 in the manual. The distribution of mustard SNP sites on chromosomes is shown in Figure 2 shown.

[0079] Example 2 Detection of genotypes of Brassica juncea 1.6K SNP molecular marker combination

[0080] This example provides a method for detecting the genotype of Brassica juncea 1.6KSNP molecular marker combination in Example 1. The steps are as follows:

[0081] (1) Extraction and quality control of genomic DNA

[0082] The sample DNA was extracted by magnetic bead method, and the quality of the DNA sample was tested. The quality test included measuring the DNA concentration with Qubit fluorescence quantification instrument and detecting the integrity of DNA with 1% agarose gel electrophoresis. The samples that passed the quality test were used for library preparation.

[0083] (2) Genotyping based on multiplex PCR and next-generation sequencing (conventional sequencing methods can also be used). The multiplex PCR and next-generation sequencing detection technology process is as follows: Figure 3 As shown in Figure 2, the technical principle of the detection is as follows: Figure 4 shown.

[0084] The test was performed using a kit (purchased from Huazhi Biotechnology Co., Ltd.), and the specific method is as follows:

[0085] A. Make the synthesized marker uniform to 100 nM and mix in equal volumes for later use;

[0086] B. Take 2 μL of the extracted qualified DNA in (1), 1 μL of the mixed labeling combination solution, 25 μL of amplification enzyme and buffer solution MIX, and 22 μL of water to construct a 50 μL reaction system;

[0087] C. Perform the first round of PCR amplification at 94°C for 10 min; enter the cycling phase: 94°C for 1 min, 60°C for 3 min, 72°C for 1 min, 40 cycles of amplification; complete the PCR reaction at 72°C for 20 min;

[0088] D. Perform the second stage of PCR amplification. Take 2 μL of the first round of PCR amplification product as the DNA substrate, 1 μL of sequencing adapter as the amplification primer, 15 μL of amplification enzyme and buffer mixture MIX and 22 μL of water to construct a 30 μL reaction system. The amplification conditions are: 94°C for 10 min; enter the cycle stage: 94°C for 1 min, 60°C for 3 min, 72°C for 1 min, 10 cycles of amplification; finally 72°C for 20 min;

[0089] E. After amplification, the fragments are purified and the samples of the 150-500 bp segment are selected by magnetic bead double selection to complete the file construction;

[0090] F. The library is quality controlled, sequenced and tested.

[0091] Example 3 Evaluation of genotyping effect of 1.6K SNP molecular marker combination

[0092] In order to verify the genotyping effect of the mustard 1.6K SNP molecular marker combination, the genotype detection method of the mustard 1.6K SNP molecular marker combination provided in Example 2 was used to detect 96 mustard materials.

[0093] Table 2

[0094]

[0095]

[0096] The detection results are shown in Table 2. It can be seen from the table that the mustard 1.6K molecular marker combination prepared by the scheme of the present invention can be accurately used to detect different varieties of mustard with a high detection rate.

[0097] Example 4 Application of the mustard 1.6K molecular marker combination in genetic diversity assessment and population structure analysis of mustard materials

[0098] The genetic distance matrix of 96 mustard materials (with known kinship) was constructed and cluster analysis was performed using the 1.6K molecular marker combination of mustard prepared in Example 1 and the 1.6K SNP molecular marker combination of mustard provided in Example 2. The results are as follows: Figure 5 As shown in the figure, it can be seen that if two samples have more clustering trees and are closer, the similarity is higher and the relationship is closer, such as samples 0-2 and 0-3 (consistent with the known results); there is an evolutionary relationship at different branch levels on the same branch tree, for example, 0-2 and 0-3 may have evolved from 0-12 (consistent with the known results, 0-2 and 0-3 evolved from 0-12). This shows that the mustard 1.6K molecular marker combination can be used to determine the relationship, evolutionary relationship, composition structure, etc. of different materials.

[0099] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A mustard SNP molecular marker combination, characterized in that: It consists of 1694 SNP molecular markers, the physical positions of which are determined based on sequence alignment of the mustard reference genome Bju.genome, and the specific site information is shown in Table 1 in the specification.

2. A primer set and / or probe for detecting the mustard SNP molecular marker combination as claimed in claim 1.

3. A liquid phase chip, characterized in that: The liquid phase chip comprises the primer set and / or probe as claimed in claim 2.

4. A kit, characterized in that: The kit comprises the primer set and / or probe according to claim 2.

5. Use of one of the mustard SNP molecular marker combination according to claim 1, the primer set and / or probe according to claim 2, the liquid phase chip according to claim 3, and the kit according to claim 4 in any of the following: (1) Identification of mustard germplasm resources; (2) Assessment of genetic diversity of Brassica juncea; (3) Mustard variety purity identification; (4) Authenticity identification of mustard varieties; (5) Construction of genetic map and QTL location of Brassica juncea; (6) Genome-wide association analysis of Brassica juncea; (7) Marker-assisted selection breeding of Brassica juncea; (8) Targeted improvement of mustard; (9) Multi-gene aggregation breeding of mustard; (10) Whole genome selection of Brassica juncea; (11) Molecular design breeding of mustard.

6. A method for detecting a combination of SNP molecular markers in Brassica juncea, characterized in that: The following steps are involved: 1) extracting genomic DNA of the mustard to be tested; 2) using the primer set and / or probe described in claim 2 to perform multiplex PCR amplification on the genomic DNA of the mustard to be tested to obtain an amplified product; 3) The genotype of the amplified product is measured by a second generation sequencing method to obtain the genotype of the mustard sample to be tested.

7. The detection method according to claim 6, characterized in that: The multiplex PCR amplification includes a first round of PCR amplification and a second round of PCR amplification; Preferably, the reaction system of the first round of PCR amplification includes: 1-3 μL DNA, 0.5-2 μL mixed marker combination solution, 20-30 μL amplification enzyme and buffer solution MIX, and water is added to make up to 50 μL; Preferably, the reaction procedure of the first round of PCR amplification is as follows: 93-95°C for 8-12 min; entering the cycling stage: 93-95°C for 0.5-2 min, 58-62°C for 2-4 min, 70-74°C for 0.5-2 min, 35-45 cycles of amplification; 70-74°C for 18-22 min.

8. The detection method according to claim 7, characterized in that: The reaction system of the second round of PCR amplification includes: 1-3 μL of the product obtained from the first round of PCR amplification, 0.5-2 μL of sequencing adapter, 10-20 μL of amplification enzyme and buffer mixture MIX, and water is added to make up to 30 μL.

9. The detection method according to claim 7, characterized in that: The reaction procedure of the second round of PCR amplification is as follows: 93-95°C for 8-12 min; Enter the cycling stage: 93-95℃ for 0.5-2min, 58-62℃ for 2-4min, 70-74℃ for 0.5-2min, 8-12 cycles of amplification; 70-74℃ for 18-22min.

10. A breeding method for mustard, comprising the following steps: using the detection method according to any one of claims 6 to 9 to detect DNA of the mustard to be detected, and selecting mustard for subsequent breeding.

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