SNP (Single Nucleotide Polymorphism) site related to soybean protein content and application thereof

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site related to soybean protein content and application thereof. The SNP site related to the soybean protein content is located at the 35708082nd site of the No.19 chromosome of a soybean genome, and the polymorphism is C or A. The invention further discloses an HRM melting curve method developed based on the SNP site and used for identifying the soybean protein content, and verification is carried out in a four-way recombinant inbred line material. A verification result shows that the SNP site found by the invention is remarkably related to the protein content character of the soybean seeds. The invention provides a theoretical basis for molecular assisted breeding of soybean protein content traits, and has important significance for cultivating new varieties of high-protein soybeans.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to SNP loci related to soybean protein content and their applications. Background Art

[0002] Soybean is one of the important food crops in China and also an important source of protein. It is rich in elements such as vegetable protein, calcium, phosphorus, iron, carotene, soybean lecithin, protease inhibitor, and plant cholesterol. Therefore, eating soybeans regularly can not only improve the immune function of the human body but also play a protective role in the cardiovascular and cerebrovascular system of people. Protein and oil content are two important traits of soybeans, and the protein content in soybean seeds accounts for about 40% of the total weight. The amino acid content of soybean protein is rich, including various essential amino acids for the human body, and it is a nutritious high-quality vegetable protein. In addition to being edible, soybean protein can also be used as raw materials for feeds, medicines, and health products, etc. Therefore, the demand for soybean protein is relatively large.

[0003] Soybean protein content is a quantitative trait and is easily affected by the environment. A large number of studies on QTL mapping of soybean protein content and components have been carried out at home and abroad. The research shows that for soybeans planted in different cold regions in Northeast China, their yield, protein content, and protein yield are significantly affected by variety genotypes and production environments. The QTLs controlling quality traits are basically scattered on 20 chromosomes, and because soybean protein content is a quantitative trait that is easily regulated by the environment and has a complex genetic basis. Therefore, using molecular marker-assisted breeding to explore genetic loci related to soybean protein content and analyze the genetic mechanism affecting protein content changes can provide more theoretical support for molecular-assisted breeding of soybean quality traits, etc.

[0004] On the premise of limited planting area and difficult improvement of unit yield, increasing the protein content of soybean seeds is a feasible way to increase the total protein output. The process of increasing soybean protein content by traditional breeding methods is relatively slow. With the gradual development of biological science and technology, traditional breeding can be combined with molecular breeding to screen excellent soybean varieties with high protein by detecting single nucleotide polymorphisms. Summary of the Invention

[0005] One object of the present invention is to provide a new use of a substance for detecting the genotype of SNP loci of a to-be-detected soybean.

[0006] The present invention provides the application of a substance for detecting the genotype of SNP loci of a to-be-detected soybean in any one of the following (a1)-(a8):

[0007] (a1) Identifying or assisting in identifying the protein content of a to-be-detected soybean;

[0008] (a2) Preparing a product for identifying or assisting in identifying the protein content of a to-be-detected soybean;

[0009] (a3) Screening or assisting in screening high-protein soybean varieties;

[0010] (a4) Preparing products for screening or assisting in screening high-protein soybean varieties;

[0011] (a5) Improvement of soybean varieties;

[0012] (a6) Preparing products for improving soybean varieties;

[0013] (a7) Soybean breeding;

[0014] (a8) Preparing products for soybean breeding.

[0015] Another object of the present invention is to provide a product, and the function of the product is any one of the following (c1)-(c4):

[0016] (c1) Identifying or assisting in identifying the protein content of a to-be-detected soybean;

[0017] (c2) Screening or assisting in screening high-protein soybean varieties;

[0018] (c3) Improvement of soybean varieties;

[0019] (c4) Soybean breeding.

[0020] The product provided by the present invention includes substances for detecting the genotype of SNP sites of a to-be-detected soybean.

[0021] Furthermore, the product further includes other reagents for fluorescence quantitative PCR and high-resolution melting curve analysis, such as DNA polymerase, fluorescent dyes, etc.

[0022] In some embodiments, the DNA polymerase is EasyTaq polymerase. The fluorescent dye is EvaGreen.

[0023] Even further, the product further includes positive controls (such as genomic DNA of soybean with Hap1 genotype, genomic DNA of soybean with Hap2 genotype, genomic DNA of soybean with Hap3 genotype) and negative controls.

[0024] The application of the above product in any one of the following (c1)-(c4) also belongs to the protection scope of the present invention:

[0025] (c1) Identifying or assisting in identifying the protein content of a to-be-detected soybean;

[0026] (c2) Screening or assisting in screening high-protein soybean varieties;

[0027] (c3) Improvement of soybean varieties;

[0028] (c4) Soybean breeding.

[0029] Any of the above-mentioned SNP sites is located at deoxyribonucleotide position 35708082 of chromosome 19 of the soybean genome.

[0030] Any of the above substances for detecting the genotype of the soybean SNP locus to be tested may be any of the following (b1)-(b3):

[0031] (b1) PCR primers for amplifying a soybean genomic DNA fragment including the SNP site;

[0032] (b2) a PCR reagent containing the PCR primers described in (b1);

[0033] (b3) A kit comprising the PCR primer described in (b1) or the PCR reagent described in (b2).

[0034] In a specific embodiment of the present invention, the PCR primers consist of the single-stranded DNA shown in sequence 1 and the single-stranded DNA shown in sequence 2.

[0035] Another object of the present invention is to provide a method for identifying or assisting in identifying the soybean protein content to be tested.

[0036] The method for identifying or assisting in identifying the soybean protein content provided by the present invention comprises the following steps: detecting the genotype of the soybean SNP site to be tested, and identifying or assisting in identifying the soybean protein content according to the genotype of the soybean SNP site to be tested; the SNP site is located at the 35708082nd deoxyribonucleotide of chromosome 19 of the soybean genome.

[0037] In the above method for identifying or assisting in identifying the soybean protein content to be tested, the genotype is Hap1 genotype, Hap2 genotype or Hap3 genotype;

[0038] The Hap1 genotype is a homozygous type in which the deoxyribonucleotide at position 35708082 of chromosome 19 of the soybean genome is C;

[0039] The Hap2 genotype is a homozygous type in which the deoxyribonucleotide at position 35708082 of chromosome 19 of the soybean genome is A;

[0040] The Hap3 genotype is a heterozygous type in which the deoxyribonucleotide at position 35708082 of chromosome 19 of the soybean genome is C and A.

[0041] In the above method for identifying or assisting in the identification of the protein content of the soybeans to be tested, the method for identifying or assisting in the identification of the protein content of the soybeans according to the genotype of the SNP locus of the soybeans to be tested may be that the protein content of the soybeans to be tested with the Hap1 genotype is higher than or candidate higher than that of the soybeans to be tested with the Hap2 genotype; the protein content of the soybeans to be tested with the Hap2 genotype is higher than or candidate higher than that of the soybeans to be tested with the Hap3 genotype.

[0042] In the above method for identifying or assisting in the identification of the protein content of the soybeans to be tested, the method for detecting the genotype of the SNP locus of the soybeans to be tested may be direct sequencing or sequencing the PCR product containing the SNP locus.

[0043] In the present invention, the method of sequencing is not limited, and it can be sequencing methods classified by various classification methods, such as first-generation sequencing (i.e., Sanger sequencing), second-generation sequencing (i.e., NGS sequencing), third-generation sequencing (i.e., long-read sequencing) classified by technology generations, and also genome sequencing, transcriptome sequencing classified by application fields, and single-end sequencing, paired-end sequencing, etc. classified by sequencing methods.

[0044] Further, the method for detecting the genotype of the SNP locus of the soybeans to be tested includes the following steps: using the genomic DNA of the soybeans to be tested as a template, performing PCR amplification with the above PCR primers to obtain a melting curve, and judging the genotype of the SNP locus of the soybeans to be tested according to the melting curve.

[0045] Still further, the method for judging the genotype of the SNP locus of the soybeans to be tested according to the melting curve is as follows: if the melting curve of the soybeans to be tested is the same as the melting curve of the soybeans with the Hap1 genotype, then the genotype of the soybeans to be tested is the Hap1 genotype; if the melting curve of the soybeans to be tested is the same as the melting curve of the soybeans with the Hap2 genotype, then the genotype of the soybeans to be tested is the Hap2 genotype; if the melting curve of the soybeans to be tested is the same as the melting curve of the soybeans with the Hap3 genotype, then the genotype of the soybeans to be tested is the Hap3 genotype.

[0046] Even further, the reaction system (10 μL) for the PCR amplification is as follows: genomic DNA (10 ng / μL) 0.4 μL, forward primer (single-stranded DNA molecule shown in Sequence 1) 0.4 μL, reverse primer (single-stranded DNA molecule shown in Sequence 2) 0.4 μL, EasyTaq polymerase 5 μL, EvaGreen 1 μL, ddH2O 2.8 μL. Among them, the final concentrations of the forward primer and the reverse primer in the reaction system are both 10 μM.

[0047] The reaction conditions for the PCR amplification are as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s. The second to fourth steps are set for 32 cycles, followed by extension at 72°C for 7 min, and then increasing the temperature by 0.2°C per minute until reaching 85°C to obtain the melting curve by fluorescence absorption.

[0048] Another object of the present invention is to provide a method for screening or assisting in screening high-protein soybean varieties.

[0049] The method for screening or assisting in screening high-protein soybean varieties provided by the present invention includes the following steps: selecting soybean varieties with the Hap1 genotype; the Hap1 genotype is a homozygous type with the 35,708,082nd deoxyribonucleotide on chromosome 19 of the soybean genome being C.

[0050] Finally, another object of the present invention is to provide a method for improving soybean varieties or soybean breeding.

[0051] The method for improving soybean varieties or soybean breeding provided by the present invention includes the following steps: selecting soybean varieties with the Hap1 genotype as parents for breeding.

[0052] In the above method for improving soybean varieties or soybean breeding, the indicators for variety improvement or breeding include protein content.

[0053] The purpose of the variety improvement or breeding includes cultivating high-protein soybean varieties. The high protein means that the protein content is greater than that of the parents.

[0054] The protein content in any of the above is the seed protein content. The seed protein content can be measured using a Foss grain analyzer.

[0055] Any of the above high-protein soybean varieties can be soybean varieties with a seed protein content greater than or equal to 39.4%.

[0056] The reference genome version number of the soybean genome in any of the above is Glycine max Wm82.a2.v1.

[0057] Any of the above soybeans can be any soybean germplasm resource, variety, strain or individual plant.

[0058] In some embodiments, the soybean is a recombinant inbred line obtained by using Kenfeng 14, Kenfeng 15, Kenfeng 19 and Heinong 48 as parents to prepare a double-cross combination (Kenfeng 14 × Kenfeng 15) × (Heinong 48 × Kenfeng 19) and adopting the single-seed descent method.

[0059] The present invention provides an SNP locus related to the protein content of soybeans. This SNP locus is located at position 35708082 on chromosome 19 of the soybean genome, and the polymorphism is C or A. The present invention also developed an HRM melting curve method for identifying the protein content of soybeans based on this SNP locus and verified it in a four-way recombinant inbred line material. The verification results show that: the SNP locus discovered by the present invention is significantly correlated with the protein content trait of soybean seeds. The protein content of the soybean material with the Hap1 genotype where the SNP locus is C is significantly higher than that of the soybean material with the Hap2 genotype where the SNP locus is A. The protein content of the soybean material with the Hap2 genotype where the SNP locus is A is significantly higher than that of the soybean material with the Hap3 genotype where the SNP locus is C and A. The present invention provides a theoretical basis for the molecular-assisted breeding of the protein content trait of soybeans and is of great significance for cultivating new soybean varieties with high protein content. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 It is a melting curve diagram for using HRM to identify different genotypes of the SNP locus. Among them, the upper diagram is the melting curve diagram of the fluorescence intensity of three haplotypes; the lower diagram is the solubility curve diagram of three haplotypes.

[0061] Figure 2 It is a comparative analysis diagram of the protein content of three haplotype soybean materials in the recombinant inbred population. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0062] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0063] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0064] Kenfeng 14 (corresponding to kenfeng14 in the literature), Kenfeng 15 (corresponding to kenfeng15 in the literature), Kenfeng 19 (corresponding to kenfeng19 in the literature), and Heinong 48 (corresponding to heinong48 in the literature) in the following examples are all recorded in the literature "[1] Li, Wen-Xia., Wang, Ping., Wang, Ping., Zhao, Hengxing., Sun, Xu.. QTL for Main Stem Node Number and Its Response to Plant Densities in 144 Soybean FW-RILs. Frontiers in plant science, 2021, 12.".

[0065] The construction method of the four-way recombinant inbred line (FW-RIL) material in the following examples refers to the method in the literature "Ning Hailong et al., Construction of a genetic map of a four-way recombinant inbred line population of soybean, Soybean Science, October 2015".

[0066] Example 1: Obtaining SNP Loci Related to Soybean Protein Content

[0067] I. Construction of a Soybean Protein Content Association Population and Measurement of Traits

[0068] 1. Test Materials

[0069] The test materials include four-way recombinant inbred line (FW-RIL) materials and associated materials.

[0070] Four-way recombinant inbred line (FW-RIL) materials: Using Kenfeng 14, Kenfeng 15, Kenfeng 19, and Heinong 48 as parents, two hybrid combinations ((Kenfeng 14 × Kenfeng 15) × (Heinong 48 × Kenfeng 19)) were prepared. The F1 generations of the two hybrid combinations were crossed to form the F2 generation. The F2 generation was self-crossed for 7 generations, and the four-way recombinant inbred line (FW-RIL) with homozygous genotypes for each was obtained through the single-seed descent method.

[0071] Associated materials: A resource library constructed from a natural population of 455 high-quality soybean germplasm resources, including 4 local varieties, 387 domestic varieties, and 44 foreign varieties.

[0072] 2. Experimental Methods

[0073] The FW-RIL was planted in the following 14 environments respectively from 2018 to 2021: In 2018, it was planted in Acheng City, Heilongjiang Province (E1-E2, E126.95°, N45.52°); in 2019, it was planted in Acheng City, Heilongjiang Province (E3-E4, E126.95°, N45.52°); in 2019, it was planted in Shuangyashan City, Heilongjiang Province (E5-E6, E131.15°, N46.64°); in 2020, it was planted in Acheng City, Heilongjiang Province (E7-E8, E126.95°, N45.52°); in 2020, it was planted in Shuangyashan City, Heilongjiang Province (E9-E10, E131.15°, N46.64°); in 2021, it was planted in Acheng City, Heilongjiang Province (E11-E12, E126.95°, N45.52°); in 2021, it was planted in Xiangyang Town, Heilongjiang Province (E13-E14, E126.68°, N45.72°).

[0074] The germplasm resource population was planted in the following 2 environments respectively from 2018 to 2020: In 2018, it was planted in Harbin, Heilongjiang Province (E1); in 2019, it was planted in Shuangyashan City, Heilongjiang Province (E2); in 2019, it was planted in Harbin (E3); in 2020, it was planted in Harbin (E4).

[0075] After maturity, an appropriate amount of seeds of each variety was selected to measure the protein content using a Foss grain analyzer. Each sample was measured 10 times, and the average value was used as the phenotypic value for QTL and GWAS mapping. According to the experimental data of the measured protein content, SAS9.2 and Excel2013 were used to calculate the average value, coefficient of variation, kurtosis, and skewness of the protein content of the four-way recombinant inbred lines, and normal distribution test and variance analysis were carried out. The absolute values of kurtosis and skewness in the 14 environments of FW-RIL were all close to 0, indicating that the protein content was normally distributed.

[0076] II. Joint mapping of QTL and GWAS for protein content trait

[0077] Based on the linkage map constructed in the previous study, for the FW-RIL population, two mapping methods, interval mapping (IM-ADD) and inclusive composite interval mapping (ICIM-ADD), were used to locate additive QTLs by the software GAPL. The scan step was set to 1.00 cM and the LOD threshold was set to 2.50. The PIN value of the ICIM-ADD method was set to 0.001. In 14 environments over 4 years, a total of 56 major QTLs with a phenotypic contribution rate greater than 10% related to protein content were detected in the four-way population. According to the population structure and LD results, GWAS analysis was performed using the R language mrMLM.GUI software package. There were five multi-locus methods in this software package: mrMLM, FASTmrMLM, FASTmrEMMA, pLARmEB, and pKWmEB to locate QTNs. In the first stage, the critical P-value of FASTmrEMMA was set to 0.005, and the critical P-value parameters of the other methods were set to 0.01. In the final stage, the critical LOD value of significant QTNs was set to 3. The kinship matrix used in the analysis process was also calculated by this R language software, and a total of 333 QTNs related to protein content were detected.

[0078] III. Obtaining SNP Loci Related to Soybean Protein Content

[0079] The 333 QTNs identified by association analysis in the germplasm population were compared with the 56 QTLs identified by linkage analysis in the recombinant inbred line population FW-RIL. Among them, there were 44 QTN loci in 7 QTL genomic regions repeatedly located under multiple methods and multiple environments. Potential candidate genes were searched at intervals of 43 kb on both sides of the QTN loci according to the LD decay distance (86 kb). And based on the re-sequencing results, gene sequence variation analysis between parents was performed on these genes to screen out genes with amino acid-changing sequence differences due to promoter or exon variations. Genes related to protein content were screened according to gene function annotation. Finally, an SNP locus related to soybean protein content was obtained. This SNP locus is located at position 35708082 on chromosome 19 of the soybean genome (reference genome version number: Glycine max Wm82.a2.v1). The polymorphism of this locus is C or A, and this locus is significantly correlated with the soybean protein content trait.

[0080] Example 2: High-Resolution Melting Curve (HRM) Method for Identifying Soybean Protein Content Developed Based on SNP Loci

[0081] 1. Primer Design

[0082] To identify the genotypes of the SNP loci significantly associated with the soybean protein content trait obtained in Example 1, molecular markers were developed based on the SNP locus at 35,708,082 bp on chromosome 19 of the soybean genome, and primer pairs were designed using Primer Premier 5 software to make the target product length 147 bp. The primer sequences are as follows:

[0083] Forward primer sequence: 5’-ACTTCTTGTCACTACAGCATT-3’ (Sequence 1).

[0084] Reverse primer sequence: 5’-CACACTTTTGGAAGCCTTGG-3’ (Sequence 2).

[0085] 2. High-resolution melting curve (HRM) method for identifying soybean protein content

[0086] Using the genomic DNA of the soybean to be tested as a template, PCR amplification was carried out using the primer pair designed in step 1.

[0087] The PCR reaction system (10 μL) is as follows: genomic DNA (10 ng / μL) 0.4 μL, forward primer 0.4 μL, reverse primer 0.4 μL, EasyTaq polymerase (colorless) (ComWin Biotech Co., Ltd., Cat: CW2965M) 5 μL, EvaGreen (Biotium, Cat: 31000) 1 μL, ddH2O 2.8 μL. The final concentrations of the forward primer and the reverse primer in the PCR reaction system are both 10 μM.

[0088] The PCR reaction conditions are as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s. The second to fourth steps are set for 32 cycles, extension at 72°C for 7 min, and then the temperature is increased by 0.2°C per minute to 85°C to obtain the melting curve while absorbing fluorescence.

[0089] Using Roche Perform HRM analysis using a real-time fluorescence quantitative PCR instrument. If the melting curve of the test soybean is the same as that of the soybean with the Hap1 genotype, then the genotype of the test soybean is the Hap1 genotype (CC homozygous); if the melting curve of the test soybean is the same as that of the soybean with the Hap2 genotype, then the genotype of the test soybean is the Hap2 genotype (AA genotype); if the melting curve of the test soybean is the same as that of the soybean with the Hap3 genotype, then the genotype of the test soybean is the Hap3 genotype (CA heterozygous). Among them, the soybean with the Hap1 genotype has an SNP locus of C, the soybean with the Hap2 genotype has an SNP locus of A; the soybean with the Hap3 genotype has SNP loci of C and A. The melting curves of the soybean with the Hap1 genotype, the soybean with the Hap2 genotype, and the soybean with the Hap3 genotype are as Figure 1 shown.

[0090] Example 3. Application of the high-resolution melting curve (HRM) method for identifying the protein content of soybeans

[0091] Test materials: 89 soybean materials from a four-way recombinant inbred line (FW-RIL) population.

[0092] Experimental method: Use the method in Example 2 to identify the genotypes of SNP loci in different test materials. Use a Foss grain analyzer to measure the protein content in the seeds of the test materials.

[0093] The detection results of the genotypes of SNP loci and the seed protein content of the test materials are shown in Table 1. The results show that among the 89 materials, 66 are soybean materials with the Hap1 genotype (CC genotype), 16 are soybean materials with the Hap2 genotype (AA genotype), and 7 are soybean materials with the Hap3 genotype (CA genotype).

[0094] Furthermore, compare the seed protein content of soybeans with different genotypes and perform a significant difference analysis. The results are as Figure 2 shown. The results show that the average protein content in the seeds of soybean materials with the Hap1 genotype is significantly higher than that of soybean materials with the Hap2 genotype and soybean materials with the Hap3 genotype, and the average protein content of soybean materials with the Hap2 genotype is significantly higher than that of soybean materials with the Hap3 genotype. Among them, the average protein content of soybean materials with the Hap1 genotype is 40.67%, the average protein content of soybean materials with the Hap2 genotype is 39.71%, and the average protein content of soybean materials with the Hap3 genotype is 37.61%. Therefore, in practical applications, the protein content of a test soybean can be identified by detecting the genotype of its SNP locus.

[0095] Table 1

[0096]

[0097]

[0098] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. Use of a substance for detecting the genotype of SNP locus of a to-be-detected soybean in any one of the following (a1)-(a8): (a1) Identifying or assisting in identifying the protein content of the to-be-detected soybean; (a2) Preparing a product for identifying or assisting in identifying the protein content of the to-be-detected soybean; (a3) Screening or assisting in screening high-protein soybean varieties; (a4) Preparing a product for screening or assisting in screening high-protein soybean varieties; (a5) Improvement of soybean varieties; (a6) Preparing a product for improvement of soybean varieties; (a7) Soybean breeding; (a8) Preparing a product for soybean breeding; The SNP locus is located at the 35708082nd deoxyribonucleotide on chromosome 19 of the soybean genome.

2. The application according to claim 1, wherein: The substance for detecting the genotype of SNP locus of the to-be-detected soybean is any one of the following (b1)-(b3): (b1) A PCR primer for amplifying a soybean genomic DNA fragment including the SNP locus; (b2) A PCR reagent containing the PCR primer described in (b1); (b3) A kit containing the PCR primer described in (b1) or the PCR reagent described in (b2).

3. The application according to claim 2, wherein: The PCR primer consists of the single-stranded DNA shown in Sequence 1 and the single-stranded DNA shown in Sequence 2.

4. A product, which includes the substance for detecting the genotype of SNP locus of the to-be-detected soybean according to any one of claims 1-3, and the function of the product is any one of the following (c1)-(c4): (c1) Identifying or assisting in identifying the protein content of the to-be-detected soybean; (c2) Screening or assisting in screening high-protein soybean varieties; (c3) Improvement of soybean varieties; (c4) Soybean breeding; The SNP locus is located at the 35708082nd deoxyribonucleotide on chromosome 19 of the soybean genome.

5. Use of the product according to claim 4 in any one of the following (c1)-(c4): (c1) Identifying or assisting in identifying the protein content of the to-be-detected soybean; (c2) Screening or assisting in screening high-protein soybean varieties; (c3) Improvement of soybean varieties; (c4) Soybean breeding.

6. A method for identifying or assisting in identifying the protein content of a to-be-detected soybean, the method includes the following steps: detecting the genotype of the SNP locus of the to-be-detected soybean, and identifying or assisting in identifying the protein content of the soybean according to the genotype of the SNP locus of the to-be-detected soybean; the SNP locus is located at the 35708082nd deoxyribonucleotide on chromosome 19 of the soybean genome.

7. The method according to claim 6, wherein: The genotype is Hap1 genotype, Hap2 genotype or Hap3 genotype; The Hap1 genotype is the homozygous type with the 35708082nd deoxyribonucleotide on chromosome 19 of the soybean genome being C; The Hap2 genotype is the homozygous type with the 35708082nd deoxyribonucleotide on chromosome 19 of the soybean genome being A; The Hap3 genotype is the heterozygous type with the 35708082nd deoxyribonucleotide on chromosome 19 of the soybean genome being C and A.

8. A method for screening or assisting in screening high-protein soybean varieties, comprising the following steps: selecting a soybean variety with the Hap1 genotype; the Hap1 genotype is a homozygous type in which the deoxyribonucleotide at position 35708082 on chromosome 19 of the soybean genome is C.

9. A method for soybean breeding, comprising the following steps: selecting a soybean variety with the Hap1 genotype as a parent for breeding; the Hap1 genotype is a homozygous type in which the deoxyribonucleotide at position 35708082 on chromosome 19 of the soybean genome is C.

10. A method for improving soybean varieties, comprising the following steps: selecting a soybean variety with the Hap1 genotype as a parent for breeding; the Hap1 genotype is a homozygous type in which the deoxyribonucleotide at position 35708082 on chromosome 19 of the soybean genome is C.

Images