DCAPS molecular marker related to soybean protein content and application of dCAPS molecular marker

Abstract

The invention discloses a dCAPS molecular marker for rapidly identifying the content of soybean protein and application of the dCAPS molecular marker. An upstream primer sequence of a primer pair for amplifying the dCAPS molecular marker is as shown in SEQ ID No. 1, and a downstream primer sequence is as shown in SEQ ID No. 2. According to the invention, the mispairing base primer is introduced to carry out PCR amplification and enzyme digestion on the to-be-detected soybean genome DNA, and the soybean protein content can be identified according to the band size of the enzyme digestion product. The dCAPS molecular marker disclosed by the invention can be used for identifying or assisting in identifying the soybean protein content, a high-protein soybean variety and a low-protein soybean variety are distinguished from the molecular level, and the dCAPS molecular marker has important significance on soybean molecular marker assisted breeding and high-protein soybean screening and cultivation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a dCAPS molecular marker related to soybean protein content and an application thereof. Background Art

[0002] Soybean [Glycine max L.] is rich in protein and can be used to make protein food as well as feed. Protein content is an important quality trait of soybean. Soy protein is a nutritious and healthy plant protein. Soy protein contains 8 kinds of amino acids needed by the human body. Eating soy protein can meet the body's daily protein needs. Soy protein is also a plant protein that is beneficial to human health. Its protein does not contain cholesterol, so it can effectively prevent cardiovascular diseases. The isoflavones it contains can also help maintain the elasticity of arterial blood vessels. It has very high nutritional value and health value. Therefore, it is widely used in meat products, dairy products, pasta products and even medical and health products. By increasing the protein content of soybeans and cultivating high-yield and high-quality soybeans, it can not only help improve people's living standards, but also promote people's physical and mental health development, and improve the economic benefits of society.

[0003] Currently, increasing the protein content of soybean varieties through genetic improvement has become one of the important ways. Among them, single nucleotide polymorphism (SNP) markers have the advantages of high efficiency, accuracy and economy, which can greatly shorten the breeding cycle and can serve as a powerful supplement to traditional breeding technology.

[0004] Molecular markers are a new type of genetic marker that has been produced and developed after morphological markers, cytological markers and biochemical markers. The types of genetic variations they reflect are very rich, not easily affected by the environment, and the stability of the DNA sequence makes molecular markers have good repeatability. Molecular marker technology can be used for germplasm identification. Single Nucleotide Polymorphism (SNP) refers to the DNA sequence polymorphism caused by the variation of a single nucleotide. As the latest generation of molecular markers, SNPs are more polymorphic and more numerous than previous molecular markers, which is conducive to analysis. With the development of high-throughput sequencing technology, the use of whole genome sequencing to screen mutations has become the most direct method to obtain the most complete genome-level base genetic variation information.

[0005] Cleaved amplified polymorphic sequence (CAPS) is a traditional molecular marker technology based on PCR. The main principle is to design primers according to the SNP located at the restriction site and perform PCR amplification. The polymorphism of the bands after restriction electrophoresis of the amplified product can determine the presence of the restriction site, thereby realizing polymorphism detection of SNP. Summary of the invention

[0006] The purpose of the present invention is to provide a dCAPS molecular marker related to soybean protein content and application thereof.

[0007] In a first aspect, the present invention claims a new use of a substance for detecting the genotype of a soybean SNP site to be tested.

[0008] The present invention claims the use of a substance for detecting the genotype of a soybean SNP site to be tested in any one of the following (a1)-(a10):

[0009] (a1) identifying or assisting in identifying the soybean protein content to be tested;

[0010] (a2) preparing a product for identifying or assisting in identifying the soybean protein content to be tested;

[0011] (a3) screening or assisting in screening high-protein soybean varieties or low-protein soybean varieties;

[0012] (a4) preparing products for screening or assisting in screening high-protein soybean varieties or low-protein soybean varieties;

[0013] (a5) distinguishing or assisting in distinguishing high-protein soybean varieties from low-protein soybean varieties;

[0014] (a6) preparing products that distinguish or assist in distinguishing high-protein soybean varieties from low-protein soybean varieties;

[0015] (a7) soybean variety improvement;

[0016] (a8) preparing products of improved soybean varieties;

[0017] (a9) soybean breeding;

[0018] (a10) preparing soybean breeding products;

[0019] In a second aspect, the present invention claims a kit, the function of which is any one of the following (b1)-(b5):

[0020] (b1) identifying or assisting in identifying the soybean protein content to be tested;

[0021] (b2) screening or assisting in screening high-protein soybean varieties or low-protein soybean varieties;

[0022] (b3) distinguishing or assisting in distinguishing high-protein soybean varieties from low-protein soybean varieties;

[0023] (b4) soybean variety improvement;

[0024] (b5) Soybean breeding.

[0025] The kit claimed in the present invention comprises a substance for detecting the genotype of the soybean SNP site to be tested.

[0026] Any of the above-mentioned SNP sites includes (is) deoxyribonucleotide No. 44269985 and No. 44269986 on chromosome 2 of the soybean genome.

[0027] Any of the above-mentioned substances for detecting the genotype of the soybean SNP site to be tested is a PCR primer for amplifying a soybean genomic DNA fragment including the SNP site.

[0028] Furthermore, the PCR primers consist of the single-stranded DNA shown in SEQ ID No.1 and the single-stranded DNA shown in SEQ ID No.2.

[0029] Furthermore, the substance for detecting the genotype of the soybean SNP site to be tested also includes restriction endonuclease DdeI.

[0030] The use of the above kit in any of the following (c1)-(c5) also falls within the scope of protection of the present invention:

[0031] (c1) identifying or assisting in identifying the soybean protein content to be tested;

[0032] (c2) screening or assisting in screening high-protein soybean varieties or low-protein soybean varieties;

[0033] (c3) distinguishing or assisting in distinguishing high-protein soybean varieties from low-protein soybean varieties;

[0034] (c4) soybean variety improvement;

[0035] (c5) Soybean breeding.

[0036] In a third aspect, the present invention claims a method for identifying or assisting in identifying the soybean protein content to be tested.

[0037] The method for identifying or assisting in identifying the protein content of soybeans to be tested claimed in the present invention is to detect whether the genotype of the soybeans to be tested is a TG genotype or a GA genotype, and determine the protein content according to the genotype of the soybeans to be tested: if the genotype of the soybeans to be tested is a TG genotype, the soybeans to be tested are a high-protein soybean variety; if the genotype of the soybeans to be tested is a GA genotype, the soybeans to be tested are a low-protein soybean variety;

[0038] The TG genotype is a homozygous type in which the 44269985th deoxyribonucleotide of chromosome 2 of the soybean genome is T and the 44269986th deoxyribonucleotide is G;

[0039] The GA genotype is a homozygous type in which the 44269985th deoxyribonucleotide of chromosome 2 of the soybean genome is G and the 44269986th deoxyribonucleotide is A.

[0040] Furthermore, the method for detecting whether the soybean genotype to be tested is a TG genotype or a GA genotype comprises the following steps: using the soybean genomic DNA to be tested as a template, using the above-mentioned PCR primers to perform PCR amplification to obtain a PCR product, using the restriction endonuclease DdeI to digest the PCR product to obtain an enzyme digestion product; and judging whether the soybean genotype to be tested is a TG genotype or a GA genotype according to the size of the enzyme digestion product.

[0041] Furthermore, the method for judging whether the soybean genotype to be tested is the TG genotype or the GA genotype according to the size of the enzyme cleavage product is as follows: the enzyme cleavage reaction product is detected by electrophoresis. If the band size of the enzyme cleavage reaction product is 527bp, the soybean genotype to be tested is the GA genotype; if the band size of the enzyme cleavage reaction product is 504bp and 23bp, the soybean genotype to be tested is the TG genotype.

[0042] Furthermore, the reaction system (10 μL) of the PCR amplification is as follows: 1 μL of genomic DNA, 1 μL of the single-stranded DNA (10 μmol / L) shown in SEQ ID No. 1, 1 μL of the single-stranded DNA (10 μmol / L) shown in SEQ ID No. 2, 12.5 μL of 2×Phanta MaxMaster Mix, ddH 2 The final concentrations of the single-stranded DNA shown in SEQ ID No. 1 and the single-stranded DNA shown in SEQ ID No. 2 in the reaction system were both 0.4 μmol / L.

[0043] The reaction conditions of the PCR amplification are as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 1 min, 35 cycles; and finally extension at 72°C for 1 min and storage at 4°C.

[0044] The enzyme digestion reaction system is as follows: 1 ng of PCR product, 1 μL of restriction endonuclease DdeI (10 U / μL), 4 μL of Cutsmart Buffer, ddH 2 HO to make up the system to 20 μL.

[0045] The reaction conditions of the enzyme digestion are as follows: 37° C. for 1 h.

[0046] In a fourth aspect, the present invention claims a method for screening or assisting in screening high-protein soybean varieties or low-protein soybean varieties.

[0047] The method for screening or assisting screening of high-protein soybean varieties claimed in the present invention comprises the steps of selecting soybean varieties of TG genotype;

[0048] The method for screening or assisting screening of low-protein soybean varieties claimed in the present invention comprises the steps of selecting soybean varieties with GA genotype;

[0049] The TG genotype is a homozygous type in which the 44269985th deoxyribonucleotide of chromosome 2 of the soybean genome is T and the 44269986th deoxyribonucleotide is G;

[0050] The GA genotype is a homozygous type in which the 44269985th deoxyribonucleotide of chromosome 2 of the soybean genome is G and the 44269986th deoxyribonucleotide is A.

[0051] In a fifth aspect, the present invention claims protection for a method for improving soybean varieties.

[0052] The method for improving soybean varieties claimed in the present invention comprises the step of breeding the high-protein soybean varieties screened according to the above method as breeding materials.

[0053] The application of any of the above methods in soybean breeding also falls within the protection scope of the present invention.

[0054] Any of the above-mentioned protein contents is the seed protein content, which can be obtained by testing with a near-infrared grain analyzer.

[0055] Any of the above-mentioned low-protein soybean varieties is a soybean variety with a protein content of less than 41%.

[0056] Any of the above-mentioned high-protein soybean varieties is a soybean variety with a protein content greater than or equal to 41%.

[0057] The reference genome version number of any of the above-mentioned soybean genomes is Glycine max Wm82.a2.v1.

[0058] Any of the above-mentioned soybeans can be any soybean germplasm resource, variety, strain or individual plant, and specifically can be a recombinant inbred line obtained by hybridizing Dongnong L13 and Henong 60 as parents and self-pollinating their progeny for five consecutive generations.

[0059] The present invention provides a dCAPS molecular marker for rapid identification of soybean protein content and its application, wherein the upstream primer sequence of the primer pair for amplifying the dCAPS molecular marker is shown as SEQ ID No.1, and the downstream primer sequence is shown as SEQ ID No.2. The present invention adopts the introduction of mismatched base primers to perform PCR amplification and enzyme digestion on the soybean genomic DNA to be tested, and the soybean protein content can be identified according to the size of the enzyme digestion product band. The dCAPS molecular marker of the present invention can be used to identify or assist in the identification of soybean protein content, distinguish high-protein soybean varieties from low-protein soybean varieties at the molecular level, and is of great significance for soybean molecular marker-assisted breeding and the screening and cultivation of high-protein soybeans.

[0060] The beneficial effects of the present invention are as follows:

[0061] 1) The present invention uses primers with mismatched bases to perform PCR amplification on the soybean genomic DNA to be tested, thereby achieving the purpose of simultaneously screening mutant soybean genomic DNA and identifying high-protein soybean varieties, reducing screening time, and improving screening efficiency and screening accuracy.

[0062] 2) The molecular markers of the present invention can be used to judge the entire soybean protein, and can quickly screen out soybean materials with high protein properties, providing a methodological basis for optimizing soybean high-protein varieties, improving soybean yield and quality, and playing an important role in assisting soybean breeding and soybean genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The electrophoresis diagram of the genotype of the mutation site identified by dCAPS molecular markers in the present invention. Lanes 1-23 are electrophoresis diagrams of DNA of different varieties in the resource population after enzyme digestion, lane 24 is the electrophoresis diagram of DNA of the parent Dongnong L13 after enzyme digestion, and lane 25 is the electrophoresis diagram of DNA of the parent Henong 60 after enzyme digestion. DETAILED DESCRIPTION

[0064] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0065] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0066] Dongnong L13 in the following examples is described in the document “Chun-miao Z, Lei D, Yu J, et al. Effects of Transgenic DREB Toybean Dongnong 50 on Diversity of Soil Nitrogen-fixing Bacteria [J]. Journal of Northeast Agricultural University (English edition), 2015, 22 (1): 1-11.”

[0067] The Henong 60 in the following examples is recorded in the document “Han Dexian, Gai Zhijia, Liu Jingqi, Zhao Guifan, Zhang Jingtao, Han Song, Ge Jingwen, Yang Dong. Research on the high-yield potential and key technologies of Henong 60 [J]. Anhui Agricultural Science Bulletin, 2014, 20(1): 29-30”.

[0068] Example 1. Obtaining mutation sites associated with soybean protein content and developing dCAPS molecular markers 1. Obtaining mutation sites associated with soybean protein content

[0069] Through the sequencing data of soybean genes in the RIL6013 population, a mutation site related to soybean protein content was found after genotypic and phenotypic analysis. It is located at positions 44269985 and 44269986 of chromosome 2 of the soybean genome (reference genome version number: Glycine max Wm82.a2.v1). The bases of the mutation site are GA or TG, and the mutation site is significantly correlated with the soybean protein content trait. Among them, the polymorphism of position 44269985 of chromosome 2 is G / T, and the polymorphism of position 44269986 of chromosome 2 is A / G.

[0070] 2. Development of dCAPS molecular markers related to soybean protein content

[0071] According to the mutation sites significantly associated with soybean protein content obtained in step 1, the restriction endonuclease DdeI was selected using the online enzyme recognition software dCAPS Finder2.0. The restriction endonuclease DdeI has a recognition sequence of C^TNAG. The primer pair sequences are designed as follows:

[0072] dCAPS-F: 5'-GTGGGAGAAAGTGATAAAAATGC T T A -3'(SEQ ID No.1);

[0073] dCAPS-R: 5'-AAAGCTTTCTTTTAAGTCATAAAGGT-3' (SEQ ID No. 2).

[0074] Among them, the underlined bases in dCAPS-F are artificially introduced mismatched bases. When the base at the mutation site is TG, the amplification product of the primer pair dCAPS-F / dCAPS-R contains the restriction endonuclease DdeI recognition sequence and can be digested by the restriction endonuclease DdeI; when the base at the mutation site is GA, the amplification product of the primer pair dCAPS-F / dCAPS-R does not contain the restriction endonuclease DdeI recognition sequence.

[0075] Example 2: Application of dCAPS molecular markers related to soybean protein content

[0076] Test materials: 25 soybean materials from the recombinant inbred line population RIL6013. The two parents of the recombinant inbred line population RIL6013 are Dongnong L13 (TG genotype, protein content 45.50%) and Henong 60 (GA genotype, protein content 38.47%). The hybrid combination: Dongnong L13×Henong 60 was prepared using these two parents, and their offspring were self-pollinated for 5 generations to achieve homozygous genotypes for each individual. Each generation was treated with single seed transmission, and finally the recombinant inbred line population RIL6013 was obtained.

[0077] 1. Genotype testing

[0078] 1. Extraction of genomic DNA

[0079] The genomic DNA of the test materials was extracted using the CTAB method. The specific steps are as follows:

[0080] 1) Add an appropriate amount of ground sample to a 2 mL centrifuge tube, add 800 μL of CTAB extract, mix well, and place in a 65°C water bath for 10 min, turning upside down every 3 min.

[0081] 2) Add 800 μL of nucleic acid extraction solution (24:1), shake for 1 min, centrifuge at 12000 rpm for 5 min, and aspirate 600 μL of the supernatant (do not aspirate other layers) and transfer to a new 1.5 mL centrifuge tube.

[0082] 3) Add an equal volume of isopropanol (pre-cooled), invert upside down about 10 times, let stand for 2 minutes, centrifuge at 12000 rpm for 5 minutes, and discard the supernatant.

[0083] 4) Add 500 μL 70% ethanol (precooled), gently blow with a pipette tip to clean the DNA flocculent precipitate (be careful not to blow the precipitate apart), centrifuge at 12000 rpm for 2 minutes, and discard the ethanol.

[0084] 5) Air-dry in a fume hood for 30 min, add 100 μL ddH 2 O dissolved.

[0085] 6) Use a UV-visible spectrophotometer to detect DNA purity and concentration and store in a -20°C refrigerator.

[0086] 2. PCR amplification and product detection

[0087] Using the genomic DNA obtained in step 1 as a template, PCR amplification was performed using the primer pair dCAPS-F / dCAPS-R to obtain a PCR amplification product.

[0088] The PCR reaction system (10 μL) was as follows: 1 μL genomic DNA, 1 μL dCAPS-F (10 μmol / L), 1 μL dCAPS-R (10 μmol / L), 12.5 μL 2× Phanta Max Master Mix (Nanjing Novogene Biotech Co., Ltd., catalog number: P515-01 / 02 / 03), ddH 2 The final concentrations of dCAPS-F and dCAPS-R in the PCR reaction system were both 0.4 μmol / L.

[0089] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 1 min, 35 cycles; and final extension at 72°C for 1 min, followed by storage at 4°C.

[0090] Take 5 μL of PCR amplification product and use 2% agarose gel electrophoresis detection. If the PCR amplification product contains only one main band with a fragment size of 527 bp, it means that the target fragment has been successfully amplified.

[0091] 3. Enzyme digestion

[0092] After verifying that the bands are correct through a gel imaging analysis system, the PCR amplification products are subjected to enzyme digestion reaction to obtain enzyme digestion reaction products.

[0093] The enzyme digestion reaction system (10 μL) was as follows: 1 ng of PCR amplification product, 1 μL of endonuclease DdeI (10 U / μL), 4 μL of Cutsmart Buffer (Bao Ri Yi Biotechnology (Beijing) Co., Ltd., catalog number: 1248A), and ddH 2 HO to make up the system to 20 μL.

[0094] The reaction conditions are: 37°C for 1 hour.

[0095] 4. Electrophoresis

[0096] The enzyme cleavage reaction products were subjected to acrylamide gel electrophoresis. The specific steps are as follows:

[0097] (1) Preparation of acrylamide gel electrophoresis reagent

[0098] 30% acrylamide (1 L): 292 g acrylamide, 4 g methylene acrylamide, and dilute to 1 L with deionized water.

[0099] 1.5M Tris-HCL (pH 8.8, 1L): Add 181.5g Tris to 800mL deionized water and stir evenly. Adjust the pH to 8.8 with hydrochloric acid and make up to 1L.

[0100] 0.5M Tris-HCl (pH 6.8, 1 L): Add 60 g of Tris to 800 mL of deionized water, adjust the pH to 6.8 with hydrochloric acid, and make up to 1 L.

[0101] 10% ammonium persulfate: Add 1g of ammonium persulfate into 10mL of deionized water until completely dissolved. Prepare before use.

[0102] 10× electrophoresis buffer (2L): 290g glycine, 60.8g Tris, 14.88g EDTA, dissolve in deionized water and make up to 2L. Dilute 10 times before use.

[0103] Gel staining solution: Add 20 μL EB substitute to 500 mL deionized water and place in a light-proof box for later use.

[0104] Preparation of separation gel: PAGE is divided into two layers of gel, the lower layer of separation gel is prepared first, and the upper layer of concentration gel is prepared after the lower layer of gel solidifies, and the corresponding spotting hole comb is inserted. After the upper layer of gel solidifies, the plate is mounted on the electrophoresis device, fixed, electrophoresis buffer is added, and the comb is removed.

[0105] Preparation of the lower layer 12% separation gel: 1.5M Tris-HCl (pH 8.8) 6.9 mL, 30% acrylamide 11.8 mL, water 8.6 mL, 10% APS 105 μL, TEMED 26 μL.

[0106] Preparation of the upper 5% stacking gel: 0.5 M Tris-HCl (pH 6.8) 1 mL, 30% acrylamide 0.8 mL, water 3.88 mL, 10% APS 31.3 μL, TEMED 6.25 μL.

[0107] (2) Electrophoresis

[0108] Use a pipette to draw 5 μL of 2000 DNA Marker and add it to the first spot well, then draw 3 μL of the enzyme cleavage reaction product and add it to the next spot well. Turn on the electrophoresis instrument and set the electrophoresis parameters: constant voltage 230 volts, electrophoresis time about 3 hours. After the electrophoresis is completed, remove the upper concentrated gel and immerse the lower separation gel in distilled water with Gelstain staining solution (staining solution 25 μL, distilled water 20 mL) in a certain order. After staining for 20 minutes, image and save the results in the gel imaging system.

[0109] Since the amplification product of the GA genotype soybean material does not contain the restriction endonuclease DdeI recognition sequence, the enzyme cleavage reaction product cannot be cleaved by DdeI, and the length of the electrophoresis detection enzyme cleavage reaction product is 527bp, while the amplification product of the TG genotype soybean material contains the restriction endonuclease DdeI recognition sequence, so the enzyme cleavage reaction product can be cleaved by DdeI, and the length of the electrophoresis detection enzyme cleavage reaction product is 504bp and 23bp. Among them, the TG genotype is a homozygous type in which the 44269985th deoxyribonucleotide of chromosome 2 of the soybean genome is T and the 44269986th deoxyribonucleotide is G; the GA genotype is a homozygous type in which the 44269985th deoxyribonucleotide of chromosome 2 of the soybean genome is G and the 44269986th deoxyribonucleotide is A.

[0110] The electrophoretic diagram of the enzyme digestion products of GA genotype soybean materials and TG genotype soybean materials is shown in Figure 1 shown.

[0111] 2. Soybean protein content detection

[0112] At maturity, a near-infrared grain analyzer was used to detect the protein content in the grains of the test materials. Varieties with a protein content of less than 41% were defined as low-protein varieties, and varieties with a protein content of greater than or equal to 41% were defined as high-protein varieties.

[0113] 3. Correlation analysis between soybean genotype and protein content

[0114] The genotype and protein content test results of the test materials are shown in Table 1, and the results show that the TG genotype soybean materials are all high-protein varieties, while the GA genotype soybean materials are all low-protein varieties, indicating that the dCAPS molecular marker in the present invention can accurately and quickly screen out soybean materials with high protein content.

[0115] Table 1. Protein content identification results and genotype detection results in soybean seeds

[0116] Product No. Variety Type genotype 1 Low protein (40.3%) GA 2 Low protein (39.5%) GA 3 Low protein (40.5%) GA 4 Low protein (38.9%) GA 5 High protein (42.2%) TG 6 High protein (42.0%) TG 7 High protein (42.7%) TG 8 Low protein (40.4%) GA 9 Low protein (38.8%) GA 10 Low protein (39.4%) GA 11 Low protein (40.2%) GA 12 Low protein (40.9%) GA 13 Low protein (39.3%) GA 14 High protein (42.8%) TG 15 Low protein (38.9%) GA 16 High protein (43.5%) TG 17 Low protein (40.0%) GA 18 Low protein (38.6%) GA 19 High protein (43.3%) TG 20 High protein (43.2%) TG 21 Low protein (38.5%) GA 22 High protein (42.2%) TG 23 Low protein (40.1%) GA 24 High protein (45.5%) TG 25 Low protein (38.47%) GA

[0117] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of a substance for detecting the genotype of a soybean SNP locus to be tested in any one of the following (a1)-(a10): (a1) identifying or assisting in identifying the soybean protein content to be tested; (a2) preparing a product for identifying or assisting in identifying the soybean protein content to be tested; (a3) screening or assisting in screening high-protein soybean varieties or low-protein soybean varieties; (a4) preparing products for screening or assisting in screening high-protein soybean varieties or low-protein soybean varieties; (a5) distinguishing or assisting in distinguishing high-protein soybean varieties from low-protein soybean varieties; (a6) preparing products that distinguish or assist in distinguishing high-protein soybean varieties from low-protein soybean varieties; (a7) soybean variety improvement; (a8) preparing products of improved soybean varieties; (a9) soybean breeding; (a10) preparing soybean breeding products; The SNP sites include deoxyribonucleotides at positions 44269985 and 44269986 of chromosome 2 of the soybean genome.

2. The use according to claim 1, characterized in that: The material for detecting the genotype of the soybean SNP site to be tested is a PCR primer for amplifying a soybean genomic DNA fragment including the SNP site.

3. The use according to claim 2, characterized in that: The PCR primers consist of the single-stranded DNA shown in SEQ ID No.1 and the single-stranded DNA shown in SEQ ID No.

2.

4. The use according to claim 3, characterized in that: The material for detecting the genotype of the soybean SNP site to be tested also includes restriction endonuclease DdeI.

5. A kit comprising the substance for detecting the genotype of a soybean SNP site to be tested according to any one of claims 1 to 4, wherein the function of the kit is any one of the following (b1) to (b5): (b1) identifying or assisting in identifying the soybean protein content to be tested; (b2) screening or assisting in screening high-protein soybean varieties or low-protein soybean varieties; (b3) distinguishing or assisting in distinguishing high-protein soybean varieties from low-protein soybean varieties; (b4) soybean variety improvement; (b5) Soybean breeding.

6. Use of the kit according to claim 5 in any one of the following (c1) to (c5): (c1) identifying or assisting in identifying the soybean protein content to be tested; (c2) screening or assisting in screening high-protein soybean varieties or low-protein soybean varieties; (c3) distinguishing or assisting in distinguishing high-protein soybean varieties from low-protein soybean varieties; (c4) soybean variety improvement; (c5) Soybean breeding.

7. A method for identifying or assisting in identifying the protein content of a soybean to be tested, comprising: detecting whether the genotype of the soybean to be tested is a TG genotype or a GA genotype, and determining the protein content according to the genotype of the soybean to be tested: if the genotype of the soybean to be tested is a TG genotype, the soybean to be tested is a high-protein soybean variety; if the genotype of the soybean to be tested is a GA genotype, the soybean to be tested is a low-protein soybean variety; The TG genotype is a homozygous type in which the 44269985th deoxyribonucleotide of chromosome 2 of the soybean genome is T and the 44269986th deoxyribonucleotide is G; The GA genotype is a homozygous type in which the 44269985th deoxyribonucleotide of chromosome 2 of the soybean genome is G and the 44269986th deoxyribonucleotide is A.

8. A method for screening or assisting in screening high-protein soybean varieties, comprising the step of selecting soybean varieties with TG genotype; The TG genotype is a homozygous type in which the 44269985th deoxyribonucleotide of chromosome 2 of the soybean genome is T and the 44269986th deoxyribonucleotide is G; Or, a method for screening or assisting screening of low-protein soybean varieties, comprising the step of selecting soybean varieties with GA genotype; The GA genotype is a homozygous type in which the 44269985th deoxyribonucleotide of chromosome 2 of the soybean genome is G and the 44269986th deoxyribonucleotide is A.

9. A method for improving soybean varieties, comprising the step of breeding the high-protein soybean varieties screened according to the method of claim 8 as breeding materials.

10. Use of the method according to any one of claims 7 to 9 in soybean breeding.

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