CN114317798B

CN114317798B - Molecular marker related to soybean protein content and application thereof

Info

Publication number: CN114317798B
Application number: CN202111572512.1A
Authority: CN
Inventors: 杨庆; 张孟臣; 刘兵强; 孟庆民; 杨春燕; 史晓蕾; 闫龙; 赵青松; 杨永庆; 王凤敏; 赵田湉
Original assignee: Institute of Grain and Oil Crops of Hebei Academy of Agriculture and Forestry Sciences
Current assignee: Institute of Grain and Oil Crops of Hebei Academy of Agriculture and Forestry Sciences
Priority date: 2021-12-21
Filing date: 2021-12-21
Publication date: 2023-03-14
Anticipated expiration: 2041-12-21
Also published as: CN114317798A

Abstract

The invention discloses a molecular marker related to soybean protein content, a specific primer pair thereof and application thereof in soybean breeding. Experiments prove that the interpretable genetic variation rate corresponding to the results of typing by using the molecular marker and the primer pair and the results of protein content determination is 11.20-39.00%, which indicates that Chr15_ 4510542A_G is an SNP molecular marker related to the content of soybean protein, can be used for identifying or assisting in identifying the content of the soybean protein, can be used for screening varieties with the content of the soybean protein, can be used for assisting in breeding by using the soybean molecular marker, and can be used for breeding and cultivating high-protein soybeans.

Description

Molecular marker related to soybean protein content and application thereof

Technical Field

The invention relates to a molecular marker related to soybean protein content and application thereof in the field of biotechnology.

Background

Soybeans (Glycine max l.) are rich in protein and can be used for producing protein food and feed. Protein content is an important quality trait of soybeans. The soybean protein is a nutritional and healthy plant protein, contains 8 amino acids required by a human body, and can meet the daily requirement of the human body on the protein by eating the soybean protein. The soybean protein is also a plant protein beneficial to human health, and the protein does not contain cholesterol, so cardiovascular diseases can be effectively prevented, and the isoflavone is also beneficial to maintaining the elasticity of arterial blood vessels and has very high nutritional value and health care value, so the soybean protein is widely applied to meat products, dairy products, flour products and even medical health care products. By increasing the protein content of the soybeans, the high-yield and high-quality soybeans are cultivated, so that the improvement of the living standard of people is facilitated, the physical and mental health development of people can be promoted, and the social economic benefit can be improved.

The soy protein content is often controlled by a linkage group of different genes, which is both genetically influenced and significantly influenced by the environment. Researches show that the protein content of the soybean varieties planted in different ecological areas of China has obvious and wide genetic variation. By deeply analyzing QTL sites related to the quality traits, a plurality of researches also show that QTL controlling the quality traits are basically dispersed on 20 chromosomes. Because the protein content is regulated and controlled by the environment and polygene regulation and control characters, the protein has a complex genetic basis. Therefore, by mining and controlling the QTL related to the soybean protein and analyzing the genetic mechanism influencing the protein content change, more theoretical supports can be provided for molecular assisted breeding of soybean quality traits and the like.

In recent decades, QTL positioning research related to soybean protein content has made great progress. However, since the change of protein content is influenced by the complex genetic basis and the external environment, the research results obtained by different researchers are very different. A number of researchers have conducted extensive studies on soybean protein content inheritance using various materials and methods, including traditional quantitative genetic experiments, molecular marker-based QTL analysis, and candidate gene prediction, among others. The detection of a population under multiple environments or different genetic backgrounds shows that a plurality of QTL sections capable of being detected exist on chromosome 6 (C2 linkage group), chromosome 15 (E linkage group) and chromosome 20 (I linkage group), and the results show that the detected QTL sections have stable influence on protein content, are possibly less influenced by the environments and can further finely position the 3 related loci (load, 2015). Bolon et al (2010) analyzed the isogenic material using a soybean gene chip, and finally defined the QTL region on chromosome 20 linkage group to within 8.4 Mb.

Therefore, genetic sites related to soybean quality traits are explored, the efficiency of improving the soybean quality by molecular marker assisted breeding is improved, and theoretical support is provided for further cultivating high-yield and high-quality soybean varieties.

Reference documents

Weihe, wangjin, lu is the nation the molecular genetic research progress of protein content of soybean seeds [ J ]. Proceedings of oil crops in China 2015,37 (03): 394-400.

Disclosure of Invention

The invention aims to solve the technical problem of accurately identifying the content of the soybean protein.

In order to solve the technical problems, the invention provides application of a primer pair in any one of A1-A3, wherein the SNP is Chr15_4510542_A _G, is positioned on chromosome 15, is the 30 th nucleotide of SEQ ID No.1, and has the nucleotide type of A or G;

a1, application in identification or auxiliary identification of the content of the soybean protein;

a2, application in preparation of products for identification or auxiliary identification of soybean protein content;

a3, application in soybean breeding or application in preparing soybean breeding products;

the primer pair is a composition consisting of P1 and P2; the P1 is a single-stranded DNA specifically combined with the upstream of the 1 st to 29 th positions of the double-stranded DNA shown in SEQ ID No. 1; and P2 is single-stranded DNA specifically combined with the downstream 148 th-168 th positions of the double-stranded DNA shown in SEQ ID No. 1.

In the application, P1 is single-stranded DNA shown in SEQ ID No.2, and P2 is single-stranded DNA shown in SEQ ID No. 3.

The application of a substance for detecting polymorphism or genotype of SNP in soybean genome in any one of A1-A3, wherein the SNP is 30 th nucleotide of SEQ ID No.1, and the type of the nucleotide is A or G;

a3, application in soybean breeding or application in preparation of soybean breeding products.

The soybean is Chr15_4510542_A _Gwhich is homozygous soybean.

In the above application, the substance is any one of the following substances:

d1 The substance contains PCR primers for amplifying a soybean genomic DNA fragment including the SNP;

d2 The substance is a PCR reagent containing the PCR primer of D1);

d3 The substance is a kit containing the PCR primer D1) or the PCR reagent D2);

d4 Said substance contains D1) said PCR primers and restriction enzyme BclI;

d5 The substance is a reagent containing the PCR reagent D2) and the restriction enzyme BclI.

In the application, the PCR primer is a primer pair consisting of P1 and P2; the P1 is a single-stranded DNA specifically combined with the upstream of the 1 st to 29 th positions of the double-stranded DNA shown in SEQ ID No. 1; and P2 is single-stranded DNA specifically combined with the downstream of the 148 th-168 th position of the double-stranded DNA shown in SEQ ID No. 1.

The invention also provides a product containing the substance for detecting the polymorphism or the genotype of the SNP in the soybean genome, wherein the product is any one of C1) -C3):

c1 Products that detect single nucleotide polymorphisms or genotypes associated with soy protein content;

c2 Product to identify or assist in identifying soy protein content;

c3 Products for soybean breeding.

The invention also provides a method for detecting the genotype of the SNP in the soybean genome.

The method for detecting the genotype of the SNP in the soybean genome provided by the invention comprises the following steps of I or II:

i, including the following K1) and K2):

k1 Taking the genome DNA of the soybean to be detected as a template, and carrying out PCR amplification by adopting the primer pair to obtain a PCR product;

k2 Detecting the PCR product obtained in the step K1), and determining the genotype of the SNP of the soybean to be detected according to the PCR product:

the genotype of the soybean to be detected, of which the 30 th nucleotide corresponding to SEQ ID No.1 is G, of the PCR product is GG; the genotype of the soybean to be detected, of which the 30 th nucleotide corresponding to SEQ ID No.1 of the PCR product is only A, is AA;

II, including the following L1) and L2):

l1) taking the DNA of a soybean genome to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of P1 and P2 to obtain a PCR product;

l2) the following L21) or L22):

l21) digesting the PCR product obtained in the step L1) with BclI enzyme, detecting the size of the digested product, and determining the genotype of the SNP of the soybean to be detected according to the size of the digested product:

the genotype of the soybean to be detected, of which the enzyme digestion product is a 168bp DNA fragment, is GG; the genotype of the soybean to be detected of which the enzyme digestion product is only 143bp DNA fragment and 25bp DNA fragment is AA;

l22) detecting the sequence of the PCR product obtained in the step L1), and determining the genotype of the SNP of the soybean to be detected according to the sequence:

the genotype of the soybean to be detected, of which the 30 th nucleotide of the PCR product corresponding to SEQ ID No.1 is G, is GG; the genotype of the soybean to be detected, of which the 30 th nucleotide of the PCR product corresponding to SEQ ID No.1 is A, is AA.

The invention also provides a method for identifying or assisting in identifying the content of the soybean protein, which comprises the steps of detecting the polymorphism of the soybean to be detected by the method for detecting the polymorphism of the SNP in the soybean genome, and identifying or assisting in identifying the content of the soybean protein according to the polymorphism of the soybean to be detected; the content of the soybean protein to be detected with the polymorphism GG is higher than or the candidate content is higher than that of the soybean protein to be detected with the polymorphism AA.

Another technical problem to be solved by the present invention is how to perform soybean breeding.

In order to solve the above technical problems, the present invention provides a method for breeding soybean, comprising detecting a polymorphism of soybean to be tested by the above method for detecting a polymorphism of SNP in soybean genome, and selecting soybean having polymorphism GG as a parent for breeding.

The invention also provides any one of the following applications A1-A3 of the primer pair:

In the application, the soybean breeding is to cultivate high-protein soybeans.

As above, the soybeans are pure lines. The soybean to be detected can be a Recombinant Inbred Line (RIL) of HJ117 (female parent) x Hobbit (male parent). The soybean to be detected can be a Recombinant Inbred Line (RIL) of HJ117 (female parent) x Zhonghuang 13 (female parent). The soybean to be detected can be a Recombinant Inbred Line (RIL) of HJ117 (female parent) x zihuang 34 (male parent).

In one embodiment of the invention, primers are used for amplifying soybean genomic DNA including SNP locus Chr15_4510542 \A _G, the reaction product is subjected to enzyme digestion by BclI, and the nucleotide type of the SNP locus is determined by performing gel electrophoresis on the enzyme digestion product. Experiments prove that whether aiming at the RIL population of HJ117 multiplied by Hobbit, the RIL population of HJ17 multiplied by Zhonghuang 13, the RIL population of HJ117 multiplied by zihuang 34 or the RIL population of HJ117 multiplied by Xudou 16, the primer disclosed by the invention is used for typing the SNP site of Chr15_ 4510542A_G at the SNP site, and the interpretable genetic variation rate corresponding to the protein content is 11.20% -39.00%, which indicates that Chr15_4510542 u A _Gis an SNP molecular marker related to the soybean protein content, can be used for identifying or assisting in identifying the soybean protein content, can be used for screening varieties with the soybean protein content, can be used for assisting in breeding by the soybean molecular marker, and can be used for breeding and cultivating high-protein soybeans.

Drawings

Fig. 1 is a GWAS analysis result graph of the protein trait of the progeny population using HJ117 as the female parent in example 1 of the present invention.

FIG. 2 is a graph showing the normal distribution of RIL population proteins and marker selection efficiency of HJ117 × Hobbit in example 2 of the present invention.

FIG. 3 is a graph showing the normal distribution of RIL population proteins and marker selection efficiency of HJ117 Xzihuang 34 in example 2 of the present invention.

FIG. 4 is a graph showing the normal distribution of RIL population proteins and the efficiency of marker selection in HJ 117X Zhonghuang 13 in example 2 of the present invention.

FIG. 5 is a graph showing the RIL population protein normal distribution and marker selection efficiency of HJ117 XXuma 16 in example 2 of the present invention.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are all conventional ones unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The soybean variety HJ117 used in the following examples was described in non-patent document "Guo Jing, xiao lei, liu Qian, zhao Qing Song, manzu, liu Bing Qiang, yanlong, wanfeng Min, zhang Meng, zhao Bao Huan, yanchun Yan.

Data were processed using SPSS11.5 statistical software and the results were expressed as mean. + -. Standard deviation and using One-way ANOVA test, P < 0.05 indicated significant differences from the control.

Example 1 obtaining of molecular markers and construction of related detection systems

1. Obtaining molecular markers

Polymorphism of different families is obtained by using soybean variety HJ117 as a female parent and hybridizing with 5-10 varieties to form RIL population material of about 1000 offspring and a simplified genome sequencing method (SLAF). The protein content of these 1000 families was further analyzed by GWAS mapping and the results are shown in the figure, co-mapping to two significant sites, where the site with the smallest P-value on chromosome 15 is Chr15_4510542, 1.46E-13, and the type of variation is a/G variation, where G is a synergistic genotype, and this functional site is named Chr15_4510542 a _g.

Chr15_4510542_A_G is a SNP site of a biallelic polymorphism in the soybean genome, located on chromosome 15, specifically at position 30 of SEQ ID No.1, and the nucleotide species is G or A (denoted by the letter R).

2. Design of molecular marker related primer

Based on the position of the SNP site, 30bp sequences are extracted on the upstream and downstream of the genome, dCAPS primers are designed according to the sequence by using a 'dCAPS Finder' program (http:// helix. Wustl. Edu/dCAPS. Html.), and single nucleotide mismatches are introduced near the SNP position so that the restriction endonuclease generates a recognition site in the amplified PCR product for one of the sequences amplified by using one of the parents as a template (Li et al, 2012).

Specific primers used for SNP site Chr15_4510542_A _Gare F and R, and the sequences are as follows:

f (front primer): 5 'TCATTCTCTCTTTGATATCAAATGCTTGATC-3' (shown as SEQ ID No. 2);

r (rear primer): 5' AGAACAAGAAATAAGGGAGC-.

The endonuclease used was BclI, the recognition site of BclI was TGATCA. When the nucleotide type of Chr15_4510542_A _Gis AA, the 24 th to 30 th positions of SEQ ID No.1 are TGATCA and can be recognized and digested by BclI; when the nucleotide species of Chr15_4510542_A _Gis GG, TGATCG at positions 24-30 of SEQ ID No.1 cannot be recognized by BclI.

3. Construction of molecular marker detection System

The construction of the molecular marker detection system is as follows:

extracting the genome DNA of the soybean to be detected, and carrying out PCR amplification by using a primer pair consisting of F and R, wherein the reaction system and the program of the PCR are shown in Table 1:

TABLE 1 PCR reaction System

Composition (I)	Amount of the composition
		10×Ex Tag HS buffer	2.5μL
0.2mM dNTP	2.0μL
		Template	1.0μL
Primer-F(10μM)	0.5μL
		Primer-R(10μM)	0.5μL
Ex Taq HS	0.2μL
		ddH ₂ O	18.3μL
Total	25μL

PCR reaction procedure: 30cycles of extension at 95 ℃ for 2min,95 ℃ for 30s,53 ℃ for 30s,72 ℃ for 10min and 16 ℃.

The amplified PCR product was detected by agarose gel electrophoresis, and the product having a single band was detected by BclI cleavage. The endonuclease systems used are shown in Table 2:

TABLE 2 enzyme digestion System

Composition (I)	Dosage of
		10×NEB Buffer	2.5μL
Restriction enzyme	1.0μL
		PCR product	10μL
ddH ₂ O	11.5μL
		Total	25μL

The digestion was carried out for 30min at the working temperature of the enzyme, and the digestion products were then detected in 3% agarose gel electrophoresis and the results were observed in a gel imaging system: the size of the band in the digestion product is 143bp smaller than that before digestion, which indicates that the genotype of the SNP locus Chr15_4510542_A_G of the soybean to be detected is AA, namely the soybean to be detected is an AA type family; the size of the band in the digestion product is consistent with that before digestion and is still 168bp, which indicates that the genotype of the SNP locus Chr15_4510542 \A_G of the soybean to be detected is GG, namely the soybean to be detected is GG type family.

Example 2 application of molecular marker-related fragments or primers to identification of Soybean protein content to be detected

To further confirm the effect in selecting high protein material for the progeny of the marker Chr15_ 4510542. 4 RIL (recombined lined) populations are randomly selected and respectively an RIL population of HJ117 multiplied by Hobbit, an RIL population of HJ17 multiplied by Zhonghuang 13, an RIL population of HJ117 multiplied by zihuang 34 and an RIL population of HJ117 multiplied by Xujiang 16 for carrying out protein content synergy verification, and the specific steps are as follows:

1. identification of protein content potentiation in the RIL population of HJ117 × Hobbit

The experimental material is that HJ117 is used as a female parent, hobbit is used as a male parent for hybridization, and then an individual of an F2 population is selected for multi-generation selfing to generate an RIL population of HJ117 x Hobbit, wherein each line in the population is relatively homozygous. 140 random families were selected from the RIL population of HJ117 × Hobbit for the following experiments:

1. determination of the protein content of the soybeans to be tested

The protein content of 140 random families was determined using a near infrared analyzer and the results are shown in column 2 of table 3.

In the RIL population of HJ117 × Hobbit, the distribution of the percentage protein content of 140 random families was fit to the normal distribution (see graph A in FIG. 2), and the variation range of the population was between 46.16 + -3.21%, indicating that the protein content is controlled by multiple genes.

2. Determination of the marker Chr15_4510542_A _G

The molecular marker detection system constructed in the third part of example 1 is used for detecting the 140 random families and the parents HJ117 and Hobbit in the RIL population of HJ117 multiplied by Hobbit, and the corresponding relation between the specific soybean protein content and the genotype of Chr15_4510542 xu A _Gis shown in Table 3:

TABLE 3 detection results of soybean molecular markers to be detected

The results of typing these families with the Chr15_4510542_A _Gmarker are shown in panel B of FIG. 2, showing that the average protein content of the AA and GG type families was 45.19% and 47.02%, respectively, the marker has an interpretable genetic variation rate of 28.41% in the population, and the average protein content of the GG type family is significantly higher than that of the AA type family (P < 0.05). Indicating that the efficiency of selecting high-protein families can be effectively improved in the population by using the marker.

2. Identification of the protein content potentiation of the RIL population of HJ117 Xzihuang 34

The experimental material is a RIL population which is produced by taking HJ117 as a female parent and taking zihuang 34 as a male parent for hybridization and then selecting individuals of the F2 population for multi-generation selfing to generate HJ117 multiplied by zihuang 34, wherein each strain in the population is relatively homozygous. The following experiments were performed in 155 random families selected from the RIL population HJ117 × zihuang 34:

1. determination of the protein content of the soybeans to be tested

Protein content of 155 random families was determined using a near infrared analyzer and the results are shown in column 2 of table 4.

In the RIL population of HJ117 Xzihuang 34, the distribution of the percentage protein content of 155 random families was fit to the normal distribution (see graph A in FIG. 3), and the variation range of the population was between 46.69 + -2.41%, indicating that the protein content was controlled by multiple genes.

2. Determination of the marker Chr15_4510542 (u A _G)

The molecular marker detection system constructed in the third part of the example 1 is used for detecting the 155 random families and the parents HJ117 and zihuang 34 in the RIL population of HJ117 multiplied by zihuang 34, and the corresponding relation of the specific soybean protein content and the polymorphism of Chr15_4510542 xu A _Gis shown in a table 4:

TABLE 4 detection results of soybean molecular markers to be detected

The results of the Chr15_4510542_A _Gmarker typing these families are shown in panel B of FIG. 3, showing that the average protein content of the AA and GG type families was 45.80% and 47.45%, respectively, and that the average protein content of the GG type family was significantly higher than that of the AA type family (P < 0.05). The interpretable genetic variation rate of the marker in the population is 34.28%, and the marker is used for effectively improving the efficiency of selecting high-protein families in the population.

3. Identification of protein content potentiation in RIL population of HJ 117X Medium yellow 13

The experimental material is a RIL population which takes HJ117 as a female parent and takes Zhonghuang 13 as a male parent for hybridization, and then individuals of the F2 population are selected for multi-generation selfing to generate HJ117 multiplied by Zhonghuang 13, and each strain in the population is relatively homozygous. 111 random families were selected from the RIL population of HJ117 × zhonghuang 13 for the following experiments:

1. determination of the protein content of the soybeans to be tested

The protein content of 111 random families was determined using a near infrared analyzer and the results are shown in column 2 of table 5.

In the RIL population HJ117 × zhonghuang 13, the distribution of the percentage protein content of 111 random families was fit to the normal distribution (see graph a in fig. 4), and the variation range of the population was between 46.66 ± 2.80%, indicating that the protein content was controlled by multiple genes.

2. Determination of the marker Chr15_4510542_A _G

The molecular marker detection system constructed in the third part of example 1 was used to detect the 111 random families and the parents HJ117 and zhonghuang 13 in the RIL population of HJ117 × zhonghuang 13, and the correspondence between the specific soybean protein content and the genotype of Chr15_4510542_a _Gis shown in table 5:

TABLE 5 detection results of soybean molecular markers to be detected

The results of typing these families with the Chr15_4510542_A _Gmarker are shown in panel B of FIG. 4, showing that the average protein content of the AA and GG type families was 46.69% and 47.29%, respectively, the interpretable genetic variation rate of this marker in the population was 11.20%, and the average protein content of the GG type family was significantly higher than that of the AA type family (P < 0.05). The marker is shown to be capable of effectively improving the efficiency of selecting high-protein families in the population.

4. Identification of protein content potentiation in RIL population of HJ117 XXudou 16

The experimental material is a RIL population which takes HJ117 as a female parent and Xujiang bean 16 as a male parent for hybridization, and then individual multi-generation selfing of the F2 population is selected to generate HJ117 XXujiang bean 16, and each strain in the population is relatively homozygous. 163 random families were selected from the RIL population of HJ117 × xu beans 16 for the following experiments:

1. determination of the protein content of the soybeans to be tested

Determination of protein content for 163 random families was determined using a near infrared analyzer and the results are shown in column 2 of table 6.

In the RIL population of HJ117 × xu bean 16, the distribution of the percentage protein content of 163 random families was fit to the normal distribution (see graph a of fig. 5), and the variation range of the population was between 46.35 ± 2.82%, indicating that the protein content was controlled by multiple genes.

2. Determination of the marker Chr15_4510542 (u A _G)

The molecular marker detection system constructed in the third part of example 1 was used to detect the 163 random families in the RIL population of HJ117 × xu beans 16, as well as the parent HJ117 and xu beans 16, and the correspondence between the specific soybean protein content and the genotype of Chr15_4510542 xu a _gis shown in table 6:

TABLE 6 detection results of soybean molecular markers to be detected

The results of typing these families with the Chr15_4510542_A _Gmarker are shown in panel B of FIG. 5, showing that the average protein content of the AA and GG type families was 45.15% and 47.35%, respectively, the interpretable genetic variation rate of this marker in the population was 39.00%, and the average protein content of the GG type family was significantly higher than that of the AA type family (P < 0.05). Indicating that the efficiency of selecting high-protein families can be effectively improved in the population by using the marker.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

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Claims

1. The application of a substance for detecting polymorphism or genotype of SNP in soybean genome in any one of A1-A3 is characterized in that the SNP is the 30 th nucleotide of SEQ ID No.1, and the nucleotide type is A or G;

a1, application in identifying the content of the soybean protein;

a2, application in preparation of products for identifying the content of the soybean protein;

a3, application in high-protein soybean breeding or application in preparation of high-protein soybean breeding products;

the soybean is selected from the group consisting of the RIL population of HJ117 × Hobbit, the RIL population of HJ117 × medium yellow 13, the RIL population of HJ117 × ziyellow 34, and the RIL population of HJ117 × xu bean 16.

2. Use according to claim 1, characterized in that: the substance is any one of the following substances:

d1 The substance contains PCR primers for amplifying soybean genomic DNA fragments including the SNP;

d2 The substance is a PCR reagent containing the PCR primer of D1);

d3 The substance is a kit containing the PCR primer D1) or the PCR reagent D2);

d4 Said substance contains D1) said PCR primers and restriction enzyme BclI;

3. Use according to claim 2, characterized in that: the PCR primer is a primer pair for amplifying a DNA fragment shown in SEQ ID No. 1.

4. Use according to claim 3, characterized in that: the primer pair consists of P1 and P2; the P1 is single-stranded DNA shown in SEQ ID No.2, and the P2 is single-stranded DNA shown in SEQ ID No. 3.

5. A method for detecting the genotype of SNP described in claim 1 in soybean genome, characterized by: is I or II as follows:

i, including the following K1) and K2):

k1 Taking the genome DNA of the soybean to be detected as a template, and carrying out PCR amplification by adopting a primer pair for amplifying the DNA segment shown in SEQ ID No.1 to obtain a PCR product;

II, including the following L1) and L2):

l1) taking the DNA of a soybean genome to be detected as a template, and carrying out PCR amplification by using a primer pair consisting of the P1 in the claim 4 and the P2 in the claim 4 to obtain a PCR product;

l2) the following L21) or L22):

the genotype of the soybean to be detected of the DNA fragment with the enzyme digestion product of 168bp is GG; the genotype of the soybean to be detected, of which the enzyme digestion products are only 143bp DNA fragments and 25bp DNA fragments, is AA;

the genotype of the soybean to be detected, of which the 30 th nucleotide corresponding to SEQ ID No.1 is G, of the PCR product is GG; the genotype of the soybean to be detected, of which the 30 th nucleotide corresponding to SEQ ID No.1 is A, of the PCR product is AA.

6. A method for identifying a soybean protein content, characterized in that the soybean protein content is identified on the basis of the genotype of the soybean to be tested by detecting the genotype of the soybean to be tested by the method of claim 5; the content of the soybean protein to be detected with the genotype of GG is higher than that of the soybean protein to be detected with the genotype of AA;

the soybeans belong to the RIL population of HJ117 × Hobbit, the RIL population of HJ117 × Zhonghuang 13, the RIL population of HJ117 × Zihuang 34, or the RIL population of HJ117 × Xujiang 16.

7. A method for breeding high-protein soybeans, which is characterized by comprising the following steps: detecting the genotype of a soybean to be tested by the method of claim 5, and selecting the soybean with the genotype of GG as a parent to breed;

the soybean belongs to the RIL population HJ117 × Hobbit, the RIL population HJ117 × medium yellow 13, the RIL population HJ117 × ziyellow 34, or the RIL population HJ117 × xu bean 16.