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

Abstract

The invention discloses an application of a dCAPS molecular marker of a soybean Glyma. 07G006300 gene in identification of a grain width. The invention provides any one of the following applications of the substance for detecting the genotype of the SNP site SW-SNP101 in the soybean genome: 1) identifying or assisting in identifying the soybean grain width character; 2) cultivating long-grain wide soybeans; the SNP site SW-SNP101 is the 101 site of the sequence 1; the genotype of the SNP site is AA or CC. Experiments prove that the molecular marker designed according to the SNP site plays an important role in researching the function of the Glyma. 07G006300 gene, can provide a theoretical basis for soybean grain width character molecule-assisted breeding, and is beneficial to cultivation of a new variety of high-yield and high-quality soybeans.

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 widely planted in the world. Soybean is generally used as a model crop in the study of leguminous crops. Grain width is an important indicator of soybean grain shape and an important trait that affects yield and appearance quality. It contributes greatly to yield in breeding. Identifying its genetic loci is helpful to increase soybean yield and cultivate high-yield varieties.

[0003] Grain width is one of the important quantitative genetic traits of soybean, 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 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:

[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 endonuclease 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 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 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 Glyma.07G006300. The 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 comprises 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 is AA at the SNP site SW-SNP101 is greater than or is a candidate greater than the soybean to be tested whose genotype is CC at the SNP site SW-SNP101;

[0035] The method shown in B comprises 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 soybean to be tested 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 method described above, 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 the set of primers described in the first aspect to perform PCR amplification to obtain a PCR product;

[0047] 3) The PCR product is digested with 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, so as to cultivate 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 found that there is natural variation in the exon region of the Glyma.07G006300 gene in soybean. At the same time, it was verified in a four-way recombinant inbred line population that the natural variation of Glyma.07G006300 will affect the grain width of soybean. Therefore, through the study of the regulation of soybean grain width by natural variation of the soybean Glyma.07G006300 gene, a theoretical basis can be provided for soybean molecular assisted breeding, which is of great significance for cultivating new varieties with high yield and high quality.

[0052] The present invention proposes an application of a soybean Glyma.07G006300 gene dCAPS molecular marker in identifying grain width. The present invention is based on a SNP site at 451458 bp of the Glyma.07G006300 gene located between 450366-456124 bp on soybean chromosome 7, where the base is C or A, and the site is significantly correlated with the soybean grain width trait. A related molecular marker is developed to identify different haplotypes of the soybean Glyma.07G006300 gene and divide it into two main haplotypes (Glyma.07G006300 and Glyma.07G006300). Hap1 、Glyma.07G006300 Hap2 ). The haplotype and seed width of Glyma.07G006300 gene in the four-way recombinant inbred line were compared. Hap2 The seed width of soybean is larger than Glyma.07G006300 Hap1 This molecular marker 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. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0054] Figure 2 This is the molecular marker used in the present invention 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 453bp.

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

[0056] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0057] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[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 reagent in the following examples is 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, 1L): Add 800mL of deionized water to 181.5g of Tris and stir evenly. Adjust the pH to 8.8 with hydrochloric acid and make up to 1L.

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

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

[0064] 10× electrophoresis buffer (2L): 290g glycine, 60.8g Tris, 14.88g EDTA, dissolve in deionized water and make up to 2L. Dilute 10 times before use.

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

[0066] Preparation of separation gel: PAGE is divided into two layers of gel, the lower layer of separation gel is prepared first, and the upper layer of concentration gel is prepared after the lower layer of gel solidifies, and the corresponding spotting hole comb is inserted. After the upper layer of gel solidifies, the plate is mounted on the electrophoresis device, fixed, electrophoresis buffer is added, and the comb is removed.

[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 its traits

[0073] Four excellent soybean varieties with large differences in grain width morphology: Kenfeng 14, Kenfeng 15, Heinong 48 and Kenfeng 19 were used as parents to prepare the double cross combination (Kenfeng 14×Kenfeng 15)×(Heinong 48×Kenfeng 19), and a four-way recombinant inbred line (FW-RIL) consisting of 144 genotype homozygous lines was obtained by single seed transmission. The related material is 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. Among them, FW-RIL was planted in 19 environments. Field trials were carried out 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, Shuangyashan (E2) and Harbin (E3) in 2019, and Harbin (E4) in 2020. After maturity, the middle 5 plants in each row were selected for indoor seed testing, and 10 representative seeds were selected to measure grain length, grain width, and grain thickness, and the average value was used as the phenotypic value for QTL and GWAS positioning. According to the experimental data of the measured grain width trait, SAS 9.2 and Excel 2013 were used to calculate the mean, coefficient of variation, kurtosis, and skewness of the 100-grain weight and grain shape (grain length, grain width, and grain thickness) of the four-way recombinant inbred lines, and normal distribution tests and variance analysis were performed. Except for E17, the absolute values ​​of kurtosis and skewness in the 19 environments of FW-RIL were close to 0, indicating that the grain width was normally distributed.

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

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

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

[0077] The 103 QTNs identified by association analysis in the germplasm population were compared with the 207 QTLs identified by linkage analysis in the recombinant inbred line population FW-RIL. Among them, 11 QTN loci were located in the genomic region of the four QTLs repeatedly located under multiple methods and multiple environments. Potential candidate genes were searched at intervals of 43kb on both sides of the QTN loci based on the LD decay distance (86kb). Based on the resequencing results, the gene sequence variation analysis between the parents of these genes was performed to screen out genes with amino acid change sequence differences caused by promoter or exon variation. Genes related to grain width were screened based on gene function annotations, and finally it was predicted that Glyma.07G006300 may be directly or indirectly related to 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·NCBItaxonomy ID: 3847. The gene is located between 450366-456124bp on soybean chromosome 7. The SNP site SW-SNP101 at the 930bp of the Glyma.07G006300 gene (sequence 4) has a base of C or A. The SNP site SW-SNP101 is the 101st position 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. The site is significantly correlated 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' (sequence 2)

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

[0084] 2. Use SNP marker primers to identify SNP locus 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, PCR amplification was performed 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 (10ng / μL) 1μL

[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: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 30 s, 32 cycles; and finally 72°C extension 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 above enzyme digestion conditions are 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 draw 5μL of 2000DNAMarker and add it to the first spotting well, then draw 3μL of the enzyme digestion product and add it to the next spotting well. Turn on the electrophoresis instrument and set the electrophoresis parameters: constant voltage 230 volts, electrophoresis time about 3 hours. After the electrophoresis is completed, remove the upper concentrated gel and immerse the lower separation gel in distilled water with Gelstain staining solution (staining solution 25ul, distilled water 20ml) in a certain order, stain for 20 minutes, and then image and save the results in the gel imaging system.

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

[0102] If the enzyme digestion product is 453bp, 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, it can be seen that the base of SNP site SW-SNP101 is C. Due to the presence of the restriction site, it can be cut by enzyme. The length of the band after enzyme cutting detected by electrophoresis is 427bp. 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 length of the electrophoresis detection band is 453bp. The haplotype of the soybean Glyma.07G006300 gene 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 genotype of SNP locus SW-SNP101 and grain width trait

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

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

[0110]

[0111]

[0112]

[0113] The above results show that the grain width of Hap2 soybeans is greater than that of Hap1 soybeans. Therefore, SNP site SW-SNP101 is 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, and 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] Or, the haplotype of soybean Glyma.07G006300 gene is detected, and the haplotype of Glyma.07G006300 gene is Glyma.07G006300 Hap2 The soybean grain width is greater than or the candidate is greater than Glyma.07G006300. The 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 (sequence 2 and sequence 3) to obtain PCR products;

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

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

[0129] Or, 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: Average soybean grain width is 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), which was consistent with the result of judging grain width by 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 implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Application of a substance for detecting the genotype of the SNP site SW-SNP101 in the soybean genome in any of the following: 1) Identify or assist in identifying soybean grain width traits; 2) Cultivate long-grain and wide-grain soybeans; The SNP site SW-SNP101 is the 101st position of sequence 1; The genotype of the SNP site is AA or CC.

2. The use according to claim 1, characterized in that: The substance is any of the following: 1) Primer set and restriction endonuclease Taq I; 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; 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); 3) A kit comprising the primer set described in 1) and restriction endonuclease Taq I.

3. Use of a substance for detecting the haplotype of soybean Glyma.07G006300 gene in any of the following: 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.

4. The use according to claim 3, characterized in that: The substance for detecting the soybean Glyma.07G006300 gene haplotype is the substance for detecting the genotype of the SNP site SW-SNP101 in the soybean genome as described in claim 1 or 2.

5. Use of the substance for detecting the genotype of the SNP site SW-SNP101 in the soybean genome as claimed in claim 1 or 2 in identifying the haplotype of the soybean Glyma.07G006300 gene; 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 Glyma.07G006300. The gene haplotype is Glyma.07G006300. Hap1 of soybeans; Glyma.07G006300 Hap2 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene is AA; Glyma.07G006300 Hap1 The genotype of the SNP site SW-SNP101 in the soybean Glyma.07G006300 gene is CC; The nucleotide sequence of the Glyma.07G006300 gene is sequence 4; The SNP site SW-SNP101 is the 101st position of sequence 1; The genotype of the SNP site is AA or CC.

6. A method for identifying or assisting in identifying soybean grain width traits, which is the following method A or B: The method shown in A comprises the following steps: 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 is AA at the SNP site SW-SNP101 is greater than or is a candidate greater than the soybean to be tested whose genotype is CC at the SNP site SW-SNP101; The method shown in B comprises 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 soybean to be tested 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.

7. The method according to claim 6, characterized in that: 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.

8. The method according to claim 6 or 7, 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 to be tested; 2) using the genomic DNA as a template and the set of primers described in claim 2 to perform PCR amplification to obtain a PCR product; 3) The PCR product is digested with 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.

9. A method for cultivating long-grain wide soybeans, comprising the following steps: Selecting soybeans with the genotype of AA at the SNP site SW-SNP101 in any one of claims 6 to 8 for cultivation, so as to cultivate long-grain and wide-grain soybeans; Or, the haplotype selected from any one of claims 6 to 8 is Glyma.07G006300 Hap2 The soybeans are cultivated to achieve the cultivation of long-grain and wide soybeans.

10. The substance for detecting the genotype of the SNP site SW-SNP101 in the soybean genome as claimed in claim 1 or 2.

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