Application of a dCAPS molecular marker of soybean Glyma.07G006300 gene in identifying grain width

Abstract

The present invention discloses an application of a dCAPS molecular marker for the soybean Glyma.07G006300 gene in identifying grain width. The present invention provides an application of a substance for detecting the genotype of the SNP site SW-SNP101 in the soybean genome in any of the following: 1) identifying or assisting in identifying the soybean grain width trait; 2) cultivating long-grain and wide soybeans; the SNP site SW-SNP101 is position 101 of sequence 1; the genotype of the SNP site is AA or CC. Experiments of the present invention have shown that the molecular marker designed by the present invention based on the SNP site plays an important role in studying the function of the Glyma.07G006300 gene, and can also provide a theoretical basis for molecular-assisted breeding of soybean grain width traits, which is conducive to the cultivation of new soybean varieties with high yield and high quality.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the application of a soybean Glyma.07G006300 gene dCAPS molecular marker in identifying grain width. Background Art

[0002] Soybean is one of the most important economic crops cultivated worldwide and is often used as a model crop in legume research. Kernel width is a key indicator of soybean kernel shape and a crucial trait influencing yield and appearance quality. It contributes significantly to yield in seed breeding, and identifying its genetic loci can help improve soybean yield and cultivate high-yielding varieties.

[0003] Grain width is one of the important quantitative genetic traits of soybeans, and improving soybean yield is inseparable from the exploration of the molecular regulatory mechanism of grain width. Summary of the Invention

[0004] The technical problem solved by the present invention is how to cultivate or detect soybeans with long grains and wide grains.

[0005] In order to solve the above technical problems, the first aspect of the present invention provides the use of a substance for detecting the genotype of the SNP site SW-SNP101 in the soybean genome in any of the following applications:

[0006] 1) Identify or assist in identifying soybean grain width traits;

[0007] 2) Cultivate long-grain and wide-grain soybeans;

[0008] The SNP site SW-SNP101 is the 101st position of sequence 1;

[0009] The genotype of the SNP site is AA or CC.

[0010] In the above application, the substance is any of the following:

[0011] 1) Primer set and restriction enzyme Taq I;

[0012] The primer set consists of a single-stranded DNA molecule or a derivative thereof shown in sequence 2, and a single-stranded DNA molecule or a derivative thereof shown in sequence 3;

[0013] 2) a PCR system containing the primer set described in 1) and an enzyme digestion system containing the restriction endonuclease Taq I described in 1);

[0014] 3) A kit comprising the primer set described in 1) and restriction endonuclease Taq I.

[0015] In a second aspect, the present invention provides the use of a substance for detecting the haplotype of the soybean Glyma.07G006300 gene in any of the following:

[0016] 1) Identify or assist in identifying soybean grain width traits;

[0017] 2) Cultivate long-grain and wide-grain soybeans;

[0018] The soybean Glyma.07G006300 gene haplotype is Glyma.07G006300 Hap1 or Glyma.07G006300 Hap2 ;

[0019] Glyma.07G006300 Hap1 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene is CC;

[0020] Glyma.07G006300 Hap2 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene is AA;

[0021] The SNP site genotype is AA or CC;

[0022] The SNP site SW-SNP101 is the 101st position in sequence 1.

[0023] The nucleotide sequence of the Glyma.07G006300 gene is sequence 4.

[0024] In the application described above, the substance for detecting the haplotype of soybean Glyma.07G006300 gene is the substance for detecting the genotype of SNP site SW-SNP101 in the soybean genome described in the first aspect.

[0025] In a third aspect, the present invention provides the use of the substance for detecting the genotype of the SNP site SW-SNP101 in the soybean genome as described in the first aspect in identifying the haplotype of the soybean Glyma.07G006300 gene;

[0026] The application is that the Glyma.07G006300 gene haplotype is Glyma.07G006300 Hap2 The soybean grain width is greater than or the candidate is greater than the Glyma.07G006300 gene haplotype is Glyma.07G006300 Hap1 of soybeans;

[0027] Glyma.07G006300 Hap2 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene is AA;

[0028] Glyma.07G006300 Hap1 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene is CC;

[0029] The nucleotide sequence of the Glyma.07G006300 gene is sequence 4;

[0030] The SNP site SW-SNP101 is the 101st position of sequence 1;

[0031] The genotype of the SNP site is AA or CC.

[0032] In a fourth aspect, the present invention provides a method for identifying or assisting in identifying the soybean grain width trait, which is the following method A or B:

[0033] The method shown in A includes the following steps:

[0034] Detecting the genotype of the SNP site SW-SNP101 in the genome of the soybean to be tested, wherein the grain width of the soybean to be tested whose genotype of the SNP site SW-SNP101 is AA is larger than or is a candidate to be larger than the soybean to be tested whose genotype of the SNP site SW-SNP101 is CC;

[0035] The method shown in B includes the following steps:

[0036] Detect the haplotype of the soybean Glyma.07G006300 gene to be tested. The haplotype is Glyma.07G006300 Hap2 The grain width of the tested soybean is greater than or the candidate greater than haplotype is Glyma.07G006300 Hap1 of soybeans to be tested;

[0037] The soybean Glyma.07G006300 gene haplotype to be tested is Glyma.07G006300 Hap2 or Glyma.07G006300 Hap1 ;

[0038] Glyma.07G006300 Hap1 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene to be tested is CC;

[0039] Glyma.07G006300 Hap2 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene to be tested is AA;

[0040] The nucleotide sequence of the Glyma.07G006300 gene is sequence 4;

[0041] The SNP site genotype is AA or CC;

[0042] The SNP site SW-SNP101 is the 101st position in sequence 1.

[0043] In the above method, the haplotype of the Glyma.07G006300 gene of the soybean to be tested is determined by detecting the genotype of the SNP site SW-SNP101 in the genome of the soybean to be tested.

[0044] In the above method, the method for detecting the genotype of the SNP site SW-SNP101 in the soybean genome to be tested is as follows:

[0045] 1) Extracting soybean genomic DNA to be tested;

[0046] 2) using the genomic DNA as a template and performing PCR amplification with the primer set described in the first aspect to obtain a PCR product;

[0047] 3) The PCR product is digested with the restriction endonuclease Taq I to obtain a digestion product. If the digestion product is 420-430 bp (the size determined by sequencing is 427) bp, the genotype of the SNP site SW-SNP101 in the soybean genome to be tested is CC. If the digestion product is 450-460 bp (the size determined by sequencing is 453) bp, the genotype of the SNP site SW-SNP101 in the soybean genome to be tested is AA.

[0048] In a fifth aspect, the present invention provides a method for cultivating long-grain wide soybeans, comprising the following steps:

[0049] Selecting soybeans with the genotype of AA at the SNP site SW-SNP101 described in the fourth aspect for cultivation to achieve the cultivation of long-grain and wide-grain soybeans;

[0050] Or, the haplotype selected in the fourth aspect is Glyma.07G006300 Hap2 The soybeans are cultivated to achieve the cultivation of long-grain and wide soybeans.

[0051] In a sixth aspect, the present invention provides a substance for detecting the genotype of the SNP site SW-SNP101 in the soybean genome as described in the first aspect. The present invention discovered that natural variation exists in the exon region of the Glyma.07G006300 gene in soybeans. Furthermore, in a four-way recombinant inbred line population, it was verified that natural variation in Glyma.07G006300 affects soybean grain width. Therefore, research on the regulation of soybean grain width by natural variation in the soybean Glyma.07G006300 gene can provide a theoretical basis for molecular-assisted soybean breeding, which is of great significance for the cultivation of new high-yield, high-quality varieties.

[0052] The present invention proposes an application of a dCAPS molecular marker of the soybean Glyma.07G006300 gene in identifying grain width. The present invention is based on a SNP site at 451458bp of the Glyma.07G006300 gene located between 450366-456124bp on soybean chromosome 7, where the base is C or A. This site is significantly correlated with the soybean grain width trait. The purpose of the present invention is to identify different haplotypes of the soybean Glyma.07G006300 gene and divide them into two main haplotypes (Glyma.07G006300 and Glyma.07G006300). Hap1 、Glyma.07G006300 Hap2 ). The haplotypes and seed widths of Glyma.07G006300 gene in the four-way recombinant inbred lines were compared. Hap2 Type soybean seeds are wider than Glyma.07G006300 Hap1 This molecular marker plays an important role in studying the function of the Glyma.07G006300 gene and can provide a theoretical basis for molecular-assisted breeding of soybean grain width traits, which is conducive to the cultivation of new high-yield and high-quality soybean varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 These are the CDS sequences of different genotypes of Glyma.07G006300 in the present invention.

[0054] Figure 2 The present invention uses molecular markers to identify different haplotypes of Glyma.07G006300. The lower band is Glyma.07G006300. Hap1 Haplotype (CC genotype), and the band size is 427bp, the upper band is Glyma.07G006300 Hap2 Haplotype (AA genotype), and the band size is 453 bp.

[0055] Figure 3This is a comparative analysis diagram of the grain width of the Glyma.07G006300 haplotype in the recombinant self-pollinated population of the present invention. DETAILED DESCRIPTION

[0056] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0057] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0058] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0059] The acrylamide gel electrophoresis reagents in the following examples were prepared as follows:

[0060] 30% acrylamide (1 L): 292 g acrylamide, 4 g methylene acrylamide, and dilute to 1 L with deionized water.

[0061] 1.5M Tris-HCl (pH 8.8, 1 L): Add 181.5 g of Tris to 800 mL of deionized water and stir thoroughly. Adjust the pH to 8.8 with hydrochloric acid and dilute to 1 L.

[0062] 0.5M Tris-HCl (pH 6.8, 1 L): To 60 g of Tris, add 800 mL of deionized water, adjust the pH to 6.8 with hydrochloric acid, and dilute to 1 L.

[0063] 10% ammonium persulfate: Add 1g of ammonium persulfate to 10mL of deionized water until completely dissolved. Prepare immediately before use.

[0064] 10× electrophoresis buffer (2 L): 290 g of glycine, 60.8 g of Tris, and 14.88 g of EDTA. Dissolve in deionized water and adjust to 2 L. Dilute 10-fold before use.

[0065] Gel staining solution: Add 20 μL of EB substitute to 500 mL of deionized water and place in a light-proof box for later use.

[0066] Preparation of the separating gel: PAGE consists of two layers, upper and lower. Prepare the lower separating gel first. After the lower gel solidifies, prepare the upper concentrating gel and insert the corresponding spotting comb. After the upper gel solidifies, place the plate in the electrophoresis apparatus, secure it, add running buffer, and remove the comb.

[0067] Preparation of the lower layer 12% separation gel:

[0068]

[0069] Preparation of the upper 5% stacking gel:

[0070]

[0071] Example 1: Obtaining Nucleotide Mutation Sites (SNPs) Significantly Associated with Soybean Grain Width

[0072] 1. Construction of soybean grain width association population and determination of traits

[0073] Using four superior soybean varieties with significant differences in kernel width morphology—Kenfeng 14, Kenfeng 15, Heinong 48, and Kenfeng 19—as parents, a double-cross combination (Kenfeng 14 × Kenfeng 15) × (Heinong 48 × Kenfeng 19) was created. Single-seed descent was used to generate a four-way recombinant inbred line (FW-RIL) consisting of 144 homozygous lines. The related material was a natural collection of 455 high-quality soybean germplasm accessions, including four local varieties, 387 domestic varieties, and 44 international varieties. The FW-RILs were cultivated in 19 environments. Field trials were conducted in Harbin (E1, E126.63°, N45.75°) and Keshan (E2, E125.64°, N48.25°) in 2015, planted in Acheng (E3-E6, E126.95°, N45.52°), Shuangcheng (E7-E10, E126.32°, N45.53°) and Northeast Agricultural University (E11-E14) in 2016, planted in Shuangcheng (E15) in 2017, in Acheng (E16) in 2018, in Acheng (E17) in 2019, in Shuangyashan (E18, E131.15°, N46.64°) in 2019, and in Harbin (E19) in 2020. The germplasm resource population was planted in Harbin (E1) in 2018, in Shuangyashan (E2) and Harbin (E3) in 2019, and in Harbin (E4) in 2020. After maturity, the middle five plants in each row were selected for indoor testing. Ten representative seeds were selected for determination of grain length, width, and thickness, and the average values ​​were used as phenotypic values ​​for QTL and GWAS mapping. Based on the experimental data for grain width, the mean, coefficient of variation, kurtosis, and skewness of 100-grain weight and grain shape (grain length, width, and thickness) of the four-way recombinant inbred lines were calculated using SAS 9.2 and Excel 2013. Normal distribution tests and analysis of variance were performed. With the exception of E17, the absolute values ​​of kurtosis and skewness were close to zero in all 19 environments of the FW-RIL, indicating a normal distribution of grain width.

[0074] 2. Joint QTL and GWAS mapping of grain width traits

[0075] Based on a linkage map constructed in previous studies, QTLs were mapped using the GAPL software using two mapping methods: interval mapping (IM-ADD) and inclusive composite interval mapping (ICIM-ADD). The genome scan step size was 1 cM, and the minimum LOD threshold was 3. In the complete interval mapping method, a marker selection probability (PIN) of 0.001 was used to control for background. A total of 207 QTLs associated with soybean grain width were detected in four populations across 19 environments over 5 years. Genome-wide association analysis was performed using the R package mrMLM.GUI to identify QTLs. This included five methods: mrMLM, FASTmrMLM, FASTmrEMMA, pLARmEB, and pKWmEB. In the first stage, the critical P value for the FASTmrEMMA method was set at 0.005, while the critical P value parameters for the other four methods were set at 0.01. In the final stage, a critical LOD value of 3 was set for significant QTLs. The GWAS identified 103 QTLs for grain width.

[0076] 3. Nucleotide mutation sites significantly associated with soybean grain width

[0077] A comparison was made between 103 QTNs identified by association analysis in the germplasm population and 207 QTLs identified by linkage analysis in the recombinant inbred line (FW-RIL) population. Eleven QTN loci were located within the genomic region of four QTLs that were reproducibly mapped using multiple methods and environments. Potential candidate genes were identified within 43 kb intervals on either side of the QTN loci based on the LD decay distance (86 kb). Sequence variation analysis of these genes between the parents was performed based on resequencing results to identify genes with amino acid sequence differences resulting from promoter or exon variants. Genes associated with grain width were then identified based on gene functional annotation, ultimately predicting that Glyma.07G006300 may be directly or indirectly associated with grain width.

[0078] The Glyma.07G006300 gene, whose gene sequence is sequence 4, is recorded in https: / / phytozome-next.jgi.doe.gov / , Glycine max Wm82.a4.v1, Phytozome genome ID: 508·NCBI taxonomy ID: 3847. This gene is located between 450366-456124 bp on soybean chromosome 7. The SNP site SW-SNP101 at 930 bp of the Glyma.07G006300 gene (sequence 4) has a base of C or A. The SNP site SW-SNP101 is position 101 of sequence 1. The base n of the SNP site SW-SNP101 is A or C. The genotype of the SNP site is CC, AA, or AC. This site is significantly associated with the soybean grain width trait.

[0079] Example 2: Development of SNP markers significantly associated with soybean grain width

[0080] 1. Design of SNP marker primers significantly associated with soybean grain width

[0081] Molecular markers were developed based on the SNP site SW-SNP101, and the primer pair sequences were designed as follows: the restriction endonuclease Taq I was selected using the online enzyme recognition software dCAPS Finder2.0, and the restriction endonuclease recognition sequence was TCGA.

[0082] Forward primer: 5'-TTGGTAGGAAATTCGCTGAT-3' (SEQ ID NO: 2)

[0083] Reverse primer: 5'-CCAACACTTTCAGCTGCCCCTTCTC-3' (SEQ ID NO: 3)

[0084] 2. Use SNP marker primers to identify SNP site genotypes

[0085] 1) Extracting genomic DNA from each soybean plant in the four-way recombinant inbred line FW-RIL;

[0086] 2) Using genomic DNA as a template, perform PCR amplification using forward sequence and reverse primers.

[0087] The PCR amplification procedure is as follows: The PCR reaction system (10 μL) is as follows:

[0088] DNA (10 ng / μL) 1 μL

[0089] Forward primer 0.5 μL

[0090] Reverse primer 0.5 μL

[0091] 2×Es Taq MasterMix(CWBIO,CW0718M)5μL

[0092] ddH2O 3μL;

[0093] The reaction conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s, for 32 cycles, followed by a final extension at 72°C for 7 min and storage at 4°C. PCR amplification products were obtained.

[0094] 3) Enzyme digestion

[0095] The PCR amplification product was digested with restriction endonuclease Taq I.

[0096] The reaction system (10 μL) of the above enzyme digestion is as follows:

[0097]

[0098] The enzyme digestion conditions are as follows: reaction at 37° C. for two hours to obtain the enzyme digestion product.

[0099] The above enzyme digestion products were subjected to acrylamide gel electrophoresis:

[0100] Use a pipette to transfer 5 μL of 2000 DNA Marker to the first well, followed by 3 μL of the digested product to the next well. Turn on the electrophoresis instrument and set the electrophoresis parameters to a constant voltage of 230 volts and a run time of approximately 3 hours. After electrophoresis, remove the top stacking gel and sequentially immerse the bottom separating gel in distilled water supplemented with Gelstain stain (25 μL of staining solution per 20 ml of distilled water) for 20 minutes. Image the gel using a gel imaging system and save the results.

[0101] If the enzyme digestion product is 427 bp, the genotype of the SNP site SW-SNP101 in the sample to be tested is CC, and the haplotype of the Glyma.07G006300 gene in the sample to be tested is Glyma.07G006300 Hap1 ;

[0102] If the enzyme digestion product is 453 bp, the genotype of the SNP site SW-SNP101 in the sample to be tested is AA, and the haplotype of the Glyma.07G006300 gene in the sample to be tested is Glyma.07G006300 Hap2 .

[0103] The above Glyma.07G006300 gene haplotype Glyma.07G006300 Hap1 The genotype of SNP site SW-SNP101 in the Glyma.07G006300 gene is CC;

[0104] The above Glyma.07G006300 gene haplotype Glyma.07G006300 Hap2 The genotype of the SNP site SW-SNP101 in the Glyma.07G006300 gene is AA.

[0105] The electrophoresis results are as follows Figure 2 As shown, the base of SNP site SW-SNP101 is C. Due to the presence of enzyme cleavage sites, it can be cleaved by enzymes. The length of the band after enzyme cleavage detected by electrophoresis is 427 bp. The haplotype of the soybean Glyma.07G006300 gene is Glyma.07G006300. Hap1 The genotype of the soybean SNP site SW-SNP101 is CC; the base of the SNP site SW-SNP101 is A, which cannot be digested by enzymes. The electrophoresis detection band length is 453bp. The soybean Glyma.07G006300 gene haplotype is Glyma.07G006300 Hap2 The genotype of the soybean SNP site SW-SNP101 is AA.

[0106] The CDS sequence of the Glyma.07G006300 gene containing the SNP site SW-SNP101 with a C base (denoted as Glyma.07G006300Hap1-CDS in the figure) and the CDS sequence of the Glyma.07G006300 gene containing the SNP site SW-SNP101 with an A base (denoted as Glyma.07G006300Hap2-CDS in the figure) are shown in part. Figure 1 shown.

[0107] 3. Association between SNP SW-SNP101 genotype and grain width

[0108] The grain width of soybeans with genotypes of AA and CC at the SNP site SW-SNP101 in 47 lines of the four-way recombinant inbred line FW-RIL identified in 2 above was correlated with their haplotypes. The results are shown in Table 1:

[0109] Table 1 shows the grain width of different genotypes

[0110]

[0111]

[0112]

[0113] The above results showed that the grain width of Hap2 soybeans was greater than that of Hap1 soybeans. Therefore, SNP site SW-SNP101 was a site significantly associated with soybean grain width.

[0114] Therefore, the soybean grain width trait can be determined by detecting the genotype of the SNP site SW-SNP101; or a method for detecting or assisting in detecting the soybean grain width trait can be established by detecting the haplotype of the soybean Glyma.07G006300 gene, as follows:

[0115] The genotype of the SNP site SW-SNP101 in soybean is detected. The grain width of soybeans whose genotype of the SNP site SW-SNP101 is AA is larger or has a potential to be larger than that of soybeans whose genotype of the SNP site SW-SNP101 is CC.

[0116] Alternatively, the soybean Glyma.07G006300 gene haplotype is detected, and the Glyma.07G006300 gene haplotype is Glyma.07G006300 Hap2 The soybean grain width is greater than or the candidate is greater than the Glyma.07G006300 gene haplotype is Glyma.07G006300 Hap1 of soybeans.

[0117] The above method for detecting the haplotype of soybean Glyma.07G006300 gene is to detect the genotype of SNP site SW-SNP101. If the genotype of SNP site SW-SNP101 of the soybean to be tested is CC, then the haplotype of Glyma.07G006300 gene of the soybean to be tested is Glyma.07G006300. Hap1 If the genotype of the SNP site SW-SNP101 of the soybean to be tested is AA, then the haplotype of the Glyma.07G006300 gene of the soybean to be tested is Glyma.07G006300 Hap2 .

[0118] The above-mentioned method for detecting the genotype of the SNP site SW-SNP101 is as follows:

[0119] 1) Extracting soybean genomic DNA to be tested;

[0120] 2) Using genomic DNA as a template, PCR amplification was performed using primers (SEQ ID NO: 2 and SEQ ID NO: 3) to obtain a PCR product;

[0121] 3) The PCR product is digested with restriction endonuclease Taq I to obtain a digestion product. If the digestion product is 427 bp, the genotype of the SNP site SW-SNP101 in the soybean genome to be tested is CC. If the digestion product is 453 bp, the genotype of the SNP site SW-SNP101 in the soybean genome to be tested is AA.

[0122] Glyma.07G006300 Hap2 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene is AA;

[0123] Glyma.07G006300 Hap1 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene is CC.

[0124] Example 3: Application of soybean grain-wide molecular markers

[0125] Four high-quality soybean varieties with significant differences in grain width and shape: Kenfeng 14, Kenfeng 15 (Glyma.07G006300 Hap1 ), Black Farmer 48 (Glyma.07G006300 Hap2 ) and Kenfeng 19 were used as parents to prepare a recombinant inbred line population of the double cross combination (Kenfeng 14×Kenfeng 15)×(Heinong 48×Kenfeng 19).

[0126] The recombinant inbred line population was identified according to the method of detecting the genotype of the SNP site SW-SNP101 to determine the grain width trait of soybean in 2 of Example 2, and the genotype of the SNP site SW-SNP101 or the Glyma.07G006300 gene haplotype of each individual plant was obtained.

[0127] Results Glyma.07G006300 Hap1 There are 60 haplotype soybeans (genotype of SNP site SW-SNP101 is CC), Glyma.07G006300 Hap2 There were 60 haplotype soybeans (genotype of SNP site SW-SNP101 was AA).

[0128] The haplotype of the Glyma.07G006300 gene is Glyma.07G006300 Hap2 The soybean grain width is greater than or the candidate is greater than the Glyma.07G006300 gene haplotype is Glyma.07G006300 Hap1 of soybeans;

[0129] Alternatively, the grain width of soybeans whose genotype at SNP site SW-SNP101 is AA is larger or may be larger than that of soybeans whose genotype at SNP site SW-SNP101 is CC.

[0130] The grain widths of different haplotype families of Glyma.07G006300 were compared. Figure 3 As shown, it can be seen that Glyma.07G006300 Hap1 Type: soybean, average grain width 6.29mm, Glyma.07G006300 Hap2 The average grain width of soybeans of different types was 6.56 mm, which was significantly different (p<0.001), consistent with the results of grain width determination based on the haplotype of Glyma.07G006300 gene.

[0131] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of a substance for detecting the haplotype of the soybean Glyma.07G006300 gene in any of the following applications: 1) Identify or assist in identifying soybean grain width traits; 2) Cultivate long-grain and wide-grain soybeans; The soybean Glyma.07G006300 gene haplotype is Glyma.07G006300 Hap1 or Glyma.07G006300 Hap2 ; Glyma.07G006300 Hap1 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene is CC; Glyma.07G006300 Hap2 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene is AA; The SNP site genotype is AA or CC; The SNP site SW-SNP101 is the 101st position of sequence 1; The nucleotide sequence of the Glyma.07G006300 gene is sequence 4.

2. The use according to claim 1, characterized in that: The substance for detecting the soybean Glyma.07G006300 gene haplotype is any one of the following: 1) Primer set and restriction enzyme Taq I; The primer set consists of a single-stranded DNA molecule shown in sequence 2 and a single-stranded DNA molecule shown in sequence 3; 2) a PCR system comprising the primer set described in 1) and an enzyme digestion system comprising the restriction endonuclease Taq I described in 1); 3) A kit containing the primer set described in 1) and the restriction endonuclease Taq I.

3. A method for identifying or assisting in identifying soybean grain width traits, comprising the following steps: Detect the haplotype of the soybean Glyma.07G006300 gene to be tested. The haplotype is Glyma.07G006300 Hap2 The grain width of the tested soybean is greater than or the candidate greater than haplotype is Glyma.07G006300 Hap1 of soybeans to be tested; The soybean Glyma.07G006300 gene haplotype to be tested is Glyma.07G006300 Hap2 or Glyma.07G006300 Hap1 ; Glyma.07G006300 Hap1 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene to be tested is CC; Glyma.07G006300 Hap2 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene to be tested is AA; The nucleotide sequence of the Glyma.07G006300 gene is sequence 4; The SNP site genotype is AA or CC; The SNP site SW-SNP101 is the 101st position in sequence 1.

4. The method according to claim 3, wherein: The haplotype of the soybean Glyma.07G006300 gene to be tested is determined by detecting the genotype of the SNP site SW-SNP101 in the soybean genome to be tested.

5. The method according to claim 4, characterized in that: The method for detecting the genotype of the SNP site SW-SNP101 in the soybean genome to be tested is as follows: 1) Extracting soybean genomic DNA; 2) using the genomic DNA as a template and performing PCR amplification with the primer set described in claim 2 to obtain a PCR product; 3) digesting the PCR product with the restriction endonuclease Taq I to obtain a digestion product. If the digestion product is 420-430 bp, the genotype of the SNP site SW-SNP101 in the soybean genome to be tested is CC. If the digestion product is 450-460 bp, the genotype of the SNP site SW-SNP101 in the soybean genome to be tested is AA.

6. A method for cultivating long-grain wide soybeans, comprising the following steps: The haplotype selected from any one of claims 3 to 5 is Glyma.07G006300 Hap2 The soybeans are cultivated to achieve the cultivation of long-grain and wide soybeans.

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