A dCAPS molecular marker related to soybean protein content and application thereof

Abstract

The application discloses a dCAPS molecular marker for rapidly identifying soybean protein content and application thereof. The sequence of the upstream primer of the primer pair for amplifying the dCAPS molecular marker is shown in SEQ ID No. 1, and the sequence of the downstream primer is shown in SEQ ID No. 2. The soybean genomic DNA to be detected is subjected to PCR amplification and enzyme cutting by using the mismatched base primer, and the soybean protein content can be identified according to the band size of the enzyme cutting product. The dCAPS molecular marker can be used for identifying or assisting in identifying the soybean protein content, and can distinguish the high-protein soybean varieties from the low-protein soybean varieties from the molecular level, and has important significance for the soybean molecular marker assisted breeding and the screening and cultivation of high-protein soybeans.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a dCAPS molecular marker related to soybean protein content and application thereof. BACKGROUND

[0002] Soybean [Glycine max L.] is rich in protein, which can be used for making protein food and also for producing feed. Protein content is an important quality trait of soybean. Soybean protein is a kind of plant protein with nutrition and health, which contains 8 kinds of amino acids needed by human body, and eating soybean protein can meet the daily protein demand of human body. Soybean protein is also a kind of plant protein beneficial to human health, which does not contain cholesterol, thus can effectively prevent cardiovascular diseases, and the contained isoflavones also help to maintain the elasticity of arterial blood vessels, and has very high nutritional value and health value, thus is widely used in meat products, dairy products, flour products and even medical and health products. By improving the protein content of soybean, cultivating high-yield and high-quality soybean can not only help to improve people's living standards, but also promote the development of people's physical and mental health, and can also improve the economic benefits of society.

[0003] At present, improving the protein content of soybean varieties through genetic improvement has become one of the important ways, among which, single nucleotide polymorphism (SNP) marker has the advantages of high efficiency, accuracy and economy, and can greatly shorten the breeding period, which can be used as a powerful supplement to traditional breeding techniques.

[0004] Molecular marker is a new genetic marker produced and developed after morphological marker, cytological marker and biochemical marker, which reflects a very rich genetic variation, is not easily affected by environment, and the stability of DNA sequence makes molecular marker have good repeatability. Molecular marker technology can be used for germplasm identification. Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphism caused by variation of a single nucleotide. As the latest generation of molecular markers, SNP is more polymorphic than the past molecular markers, and has more number, which is beneficial to analysis. With the development of high-throughput sequencing technology, screening mutations by using whole genome sequencing has become the most direct method and can obtain the most complete base genetic variation information at genome level.

[0005] The cleaved amplified polymorphic sequence (CAPS) technology is a traditional PCR-based molecular marker technology, and the main principle is to design primers according to SNPs located on the enzyme cutting site and perform PCR amplification, and the presence or absence of the enzyme cutting site can be determined by the polymorphism of the band after the amplified product is cut and electrophoresed, so as to realize polymorphism detection of the SNP. SUMMARY

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

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

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

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

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

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

[0012] (a4) preparing a product 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 or low-protein soybean varieties;

[0014] (a6) preparing a product for distinguishing or assisting in distinguishing high-protein soybean varieties or low-protein soybean varieties;

[0015] (a7) soybean variety improvement;

[0016] (a8) preparing a product for soybean variety improvement;

[0017] (a9) soybean breeding;

[0018] (a10) preparing a product for soybean breeding;

[0019] In the second aspect, the present application claims a kit, and the functions of the kit are any one of (b1)-(b5) as follows:

[0020] (b1) identifying or assisting in identifying the protein content of a soybean 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 or low-protein soybean varieties;

[0023] (b4) soybean variety improvement;

[0024] (b5) soybean breeding.

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

[0026] Any of the above-mentioned SNP sites comprises deoxyribonucleotides at positions 44269985 and 44269986 of chromosome 2 of the soybean genome.

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

[0028] Further, the PCR primer consists of a single-stranded DNA as shown in SEQ ID No. 1 and a single-stranded DNA as shown in SEQ ID No. 2.

[0029] Still further, the substance for detecting the genotype of a SNP site of a soybean to be tested further comprises a restriction enzyme Ddel.

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

[0031] (c1) identifying or assisting in identifying the protein content of a soybean 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 or low-protein soybean varieties;

[0034] (c4) soybean variety improvement;

[0035] (c5) soybean breeding.

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

[0037] The method for identifying or assisting in identifying the protein content of a soybean to be tested claimed in the present application is 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.

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

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

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

[0041] Further, the method for judging whether the genotype of the soybean to be tested is the TG genotype or the GA genotype according to the size of the digestion product is as follows: performing electrophoresis detection on the digestion reaction product, if the size of the digestion reaction product band is 527 bp, then the genotype of the soybean to be tested is the GA genotype; and if the size of the digestion reaction product band is 504 bp and 23 bp, then the genotype of the soybean to be tested is the TG genotype.

[0042] Further, the reaction system (10 μL) of the PCR amplification is as follows: 1 μL of genomic DNA, 1 μL of single-stranded DNA shown in SEQ ID No. 1 (10 μmol / L), 1 μL of single-stranded DNA shown in SEQ ID No. 2 (10 μmol / L), 12.5 μL of 2×Phanta Max Master Mix, and 9.5 μL of ddH2O. The final concentration 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 is 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; final extension at 72 °C for 1 min, and preservation at 4 °C.

[0044] The reaction system of the digestion is as follows: 1 ng of PCR product, 1 μL of restriction enzyme Ddel (10 U / μL), 4 μL of Cutsmart Buffer, and ddH2O to make up the system to 20 μL.

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

[0046] In a fourth aspect, the present application 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 in screening high-protein soybean varieties claimed by the present application comprises the step of selecting soybean varieties with TG genotype;

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

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

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

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

[0052] The method for improving soybean varieties claimed by the present application 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 application.

[0054] The protein content in any of the above methods is the seed protein content, which can be specifically tested and obtained by using a near-infrared grain analyzer.

[0055] The low-protein soybean variety in any of the above methods is a soybean variety with a protein content of less than 41%.

[0056] The high-protein soybean variety in any of the above methods is a soybean variety with a protein content of greater than or equal to 41%.

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

[0058] The soybean in any of the above methods can be any soybean germplasm resource, variety, strain or single plant, and can be specifically a recombinant inbred line obtained by crossing Dongnong L13 and He'nong 60 as parents and then continuously selfing the offspring for 5 generations.

[0059] The application provides a dCAPS molecular marker for rapidly identifying soybean protein content and application thereof, wherein the sequence of the upstream primer of the primer pair for amplifying the dCAPS molecular marker is shown as SEQ ID No. 1, and the sequence of the downstream primer is shown as SEQ ID No. 2. The soybean protein content can be identified according to the size of the enzyme digestion product band by adopting the mismatched base primer pair to perform PCR amplification on the soybean genomic DNA to be tested and enzyme digestion. The dCAPS molecular marker can be used for identifying or assisting in identifying the soybean protein content, and distinguishes the high-protein soybean varieties and the low-protein soybean varieties from the molecular level, which has important significance for the soybean molecular marker assisted breeding and screening and cultivation of high-protein soybeans.

[0060] The application has the following beneficial effects:

[0061] 1) The application adopts the mismatched base primer pair to perform PCR amplification on the soybean genomic DNA to be tested, realizes the purpose of simultaneously screening the mutant soybean genomic DNA and identifying the high-protein soybean varieties, reduces the screening time, and improves the screening efficiency and screening accuracy.

[0062] 2) The molecular marker can judge the whole soybean protein, can quickly screen the soybean material with high protein properties, provides a method basis for the optimized high-protein soybean varieties, improves the soybean yield and quality, and has important roles in assisting soybean breeding and genetic improvement of soybeans. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 It is an electrophoresis map for identifying the genotype of the mutant site by the dCAPS molecular marker in the application. Among them, lanes 1-23 are the electrophoresis maps of the DNA enzyme digestion of different varieties in the resource population, lane 24 is the DNA enzyme digestion map of the parent Dongnong L13, and lane 25 is the DNA enzyme digestion map of the parent He'nan 60. DETAILED DESCRIPTION

[0064] The application will be further described in detail in combination with specific embodiments. The examples provided below are only for illustrating the application, and are not intended to limit the scope of the application. The examples provided below can be used as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application.

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

[0066] Dongnong L13 in the following examples is described in the literature“Chun-miao Z, Lei D, Yu J, et al. Effects of Transgenic DREB Toybean Dongnong50 on Diversity of Soil Nitrogen-fixing Bacteria[J]. Journal of Northeast Agricultural University (English edition), 2015, 22(1): 1-11.”.

[0067] HeNong 60 in the following examples is described in the literature“Han Dexian, Gai Zhijia, Liu Jingqi, Zhao Guifan, Zhang Jingtao, Han Song, Ge Jingwen, Yang Dong. HeNong 60 high yield potential and key technology research [J]. Anhui Agricultural Bulletin, 2014, 20(1): 29-30”.

[0068] Example 1, obtaining of mutation site related to soybean protein content and development of dCAPS molecular marker

[0069] Through the soybean gene sequencing data of RIL6013 population, a mutation site related to soybean protein content was found after genotypic and phenotypic analysis, which was located at the 44269985th and 44269986th sites of chromosome 2 of soybean genome (reference genome version number: Glycine max Wm82.a2.v1), the base of the mutation site was GA or TG, and the mutation site was significantly related to soybean protein content. Among them, the polymorphism of the 44269985th site of chromosome 2 was G / T, and the polymorphism of the 44269986th site of chromosome 2 was A / G.

[0070] Two, development of dCAPS molecular marker related to soybean protein content

[0071] According to the mutation site obtained in step one which is significantly related to soybean protein content, the online enzyme recognition software dCAPS Finder2.0 is used to select the restriction endonuclease DdeI, the enzyme digestion recognition sequence of the restriction endonuclease DdeI is C^TNAG, and the primer pair sequence is 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] In the dCAPS-F, the base indicated by underline is a mismatch base introduced artificially. When the base at the mutation site is TG, the amplification product of the primer pair dCAPS-F / dCAPS-R contains a restriction endonuclease Ddel recognition sequence, which can be digested by the restriction endonuclease Ddel; and when the base at the mutation site is GA, the amplification product of the primer pair dCAPS-F / dCAPS-R does not contain a restriction endonuclease Ddel recognition sequence.

[0075] Example 2, Application of dCAPS molecular marker related to soybean protein content

[0076] Test materials: 25 soybean materials derived 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 He'nan 60 (GA genotype, protein content 38.47%). The two parents are used to prepare a cross combination: Dongnong L13 x He'nan 60, and the offspring is continuously self-crossed for 5 generations to reach the homozygous genotype of each individual. Single seed descent method is used for each generation, and finally the recombinant inbred line population RIL6013 is obtained.

[0077] I. Genotype detection

[0078] 1. Extraction of genomic DNA

[0079] The genomic DNA of the test material is extracted by CTAB method, and 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 extraction solution, mix well, and place in a 65°C water bath for 10 min, flip up and down every 3 min.

[0081] 2) Add 800 μL of nucleic acid extraction solution (24:1), shake for 1 min, then centrifuge at 12000 rpm for 5 min, and transfer 600 μL of supernatant (without other layers) to a new 1.5 mL centrifuge tube.

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

[0083] 4) Add 500 μL of 70% ethanol (pre-cooled), gently blow the DNA flocculent precipitate with a gun head (note that the precipitate should not be blown apart), centrifuge at 12000 rpm for 2 min, and discard the ethanol.

[0084] 5) Air dry in the fume hood for 30 min, add 100 μL ddH2O to dissolve.

[0085] 6) Use ultraviolet visible spectrophotometer to detect DNA purity and concentration, and store in -20℃ refrigerator.

[0086] 2. PCR amplification and product detection

[0087] The genomic DNA obtained in step 1 is used as a template, and primer pair dCAPS-F / dCAPS-R is used for PCR amplification to obtain a PCR amplification product.

[0088] The PCR reaction system (10 μL) is as follows: genomic DNA 1 μL, dCAPS-F (10 μmol / L) 1 μL, dCAPS-R (10 μmol / L) 1 μL, 2x Phanta Max Master Mix (Nanjing Novogene Bio-tech Co., Ltd., Catalog No.: P515-01 / 02 / 03) 12.5 μL, ddH2O 9.5 μL. The final concentration of dCAPS-F and dCAPS-R in the PCR reaction system is 0.4 μmol / L.

[0089] The PCR reaction conditions are as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles; finally 72℃ extension for 1 min, 4℃ storage.

[0090] 5 μL of the PCR amplification product is taken and subjected to electrophoresis detection using 2% agarose gel. If the PCR amplification product contains only one main band with a fragment size of 527 bp, it indicates that the target fragment is successfully amplified.

[0091] 3. Enzymatic digestion

[0092] After verifying the correct band by gel imaging analysis system, the PCR amplification product is subjected to enzymatic digestion reaction to obtain an enzymatic digestion product.

[0093] The enzymatic digestion reaction system (10 μL) is as follows: PCR amplification product 1 ng, endonuclease DdeI (10 U / μL) 1 μL, Cutsmart Buffer (Bodipy Biotech (Beijing) Co., Ltd., Catalog No.: 1248A) 4 μL, ddH2O to make up the system to 20 μL.

[0094] The reaction conditions are as follows: 37℃ reaction for 1 h.

[0095] 4. Electrophoresis

[0096] The enzymatic digestion product is subjected to acrylamide gel electrophoresis. The specific steps are as follows:

[0097] (1) Preparation of reagents for acrylamide gel electrophoresis

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

[0099] 1.5 M Tris-HCl (pH 8.8, 1 L): Tris 181.5 g, add 800 mL deionized water, stir well, adjust pH to 8.8 with hydrochloric acid, and dilute to 1 L.

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

[0101] 10% ammonium persulfate: ammonium persulfate 1 g, add 10 mL deionized water to completely dissolve, and prepare as needed.

[0102] 10x electrophoresis buffer (2 L): glycine 290 g, Tris 60.8 g, EDTA 14.88 g, dissolve in deionized water and dilute to 2 L, and dilute 10 times when used.

[0103] Gel staining solution: add 20 μL EB substitute to 500 mL deionized water, and store in a light-proof box.

[0104] Preparation of separation gel: PAGE is divided into upper and lower gels, first prepare the lower separation gel, and then prepare the upper concentrated gel after the lower gel solidifies, and insert the corresponding sample hole comb. After the upper gel solidifies, assemble the plate on the electrophoresis device, fix, add electrophoresis buffer, and remove the comb.

[0105] Preparation of lower 12% separation gel: 1.5 M 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 upper 5% concentrated 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] 5 μL 2000 DNA Marker is added to the first sample hole by using a pipette, and then 3 μL of the enzyme digestion product is added to the subsequent sample hole. The switch of the electrophoresis instrument is turned on, and the electrophoresis parameters are set: constant voltage 230 volts, and the electrophoresis time is about 3 hours. After the electrophoresis is completed, the upper concentrated gel is cut off, the lower separation gel is immersed in distilled water (25 μL of Gelstain dyeing solution and 20 mL of distilled water) in a certain order, and after being dyed for 20 min, the gel imaging system is imaged and the results are saved.

[0109] Since the GA genotype soybean material amplification product does not contain the restriction endonuclease DdeI recognition sequence, the enzyme digestion product cannot be digested by DdeI, and the band length of the enzyme digestion product detected by electrophoresis is 527 bp, while the TG genotype soybean material amplification product contains the restriction endonuclease DdeI recognition sequence, so the enzyme digestion product can be digested by DdeI, and the band length of the enzyme digestion product detected by electrophoresis is 504 bp and 23 bp. Among them, the TG genotype is the homozygous type of the deoxyribonucleotide at position 44269985 of chromosome 2 of the soybean genome being T and the deoxyribonucleotide at position 44269986 being G; the GA genotype is the homozygous type of the deoxyribonucleotide at position 44269985 of chromosome 2 of the soybean genome being G and the deoxyribonucleotide at position 44269986 being A.

[0110] The electrophoretogram of the enzyme digestion product of the GA genotype soybean material and the TG genotype soybean material is shown in Figure 1 .

[0111] Second, detection of soybean protein content

[0112] At the mature stage, the protein content in the seeds of the test materials is detected by using a near-infrared grain analyzer, and the variety type with a protein content less than 41% is defined as a low-protein variety type, and the variety type with a protein content greater than or equal to 41% is defined as a high-protein variety type.

[0113] Third, correlation analysis of soybean genotype and protein content

[0114] The genotype and protein content detection results of the test materials are shown in Table 1, and the results show that the TG genotype soybean materials are all high-protein variety types, and the GA genotype soybean materials are all low-protein variety types. It is explained that the dCAPS molecular marker in the present application can accurately and quickly screen high-protein content soybean materials.

[0115] Table 1, identification results of protein content in soybean seeds and genotype detection results

[0116] Variety 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 application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. Use of a substance for detecting genotype of a SNP site of a soybean in any one of (a1) - (a6) below: (a1) screening or assisting in screening of high-protein soybean varieties or low-protein soybean varieties; (a2) preparing a product for screening or assisting in screening of high-protein soybean varieties or low-protein soybean varieties; (a3) distinguishing or assisting in distinguishing high-protein soybean varieties or low-protein soybean varieties; (a4) preparing a product for distinguishing or assisting in distinguishing high-protein soybean varieties or low-protein soybean varieties; (a5) soybean variety improvement; (a6) soybean breeding; the SNP site comprises deoxyribonucleotide at position 44269985 and deoxyribonucleotide at position 44269986 of chromosome 2 of soybean genome; The substance for detecting the genotype of the SNP site of the soybean to be tested comprises PCR primers and a restriction endonuclease for amplifying a soybean genomic DNA fragment comprising the SNP site Dde I; the PCR primer consists of single-stranded DNA as shown in SEQ ID No. 1 and single-stranded DNA as shown in SEQ ID No.

2.

2. Use of a kit in any one of (c1) - (c4) below: (c1) screening or assisting in screening of high-protein soybean varieties or low-protein soybean varieties; (c2) distinguishing or assisting in distinguishing high-protein soybean varieties or low-protein soybean varieties; (c3) soybean variety improvement; (c4) soybean breeding; the kit comprises the substance for detecting genotype of a SNP site of a soybean as claimed in claim 1.

3. A method for screening or assisting in screening of high-protein soybean varieties, comprising the step of selecting a soybean variety with TG genotype; the TG genotype is homozygous type with T at deoxyribonucleotide at position 44269985 and G at deoxyribonucleotide at position 44269986 of chromosome 2 of soybean genome.

4. A method for screening or assisting in screening of low-protein soybean varieties, comprising the step of selecting a soybean variety with GA genotype; the GA genotype is homozygous type with G at deoxyribonucleotide at position 44269985 and A at deoxyribonucleotide at position 44269986 of chromosome 2 of soybean genome.

5. A method for soybean variety improvement, comprising the step of breeding high-protein soybean varieties screened according to the method of claim 3 as breeding materials.

6. Use of the method of any one of claims 3 - 5 in soybean breeding.

Images