CN108823330A - A kind of soybean HRM-SNP molecular labeling point labeling method and its application - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a soybean HRM-SNP molecular marker point marker and its application for the development of soybean SNP markers, the extraction of Zhexian No. No. 9 SNP typing based on HRM technology, Zhexian No. 9 HRM typing verification, construction of Zhexian No. 9 SNP fingerprint. The present invention has high throughput and short analysis time: the analysis of 96 or 384 samples can be completed at one time, which is suitable for large-scale development and detection of SNP sites; the present invention is easy to operate and does not require sequence-specific probes and subsequent sequencing. It only needs to design PCR primers, carry out PCR reaction, and then directly perform HRM to complete the genotype analysis of the sample; the invention has good specificity: the PCR product does not need subsequent treatment, and the operation is completely closed.

Description

A kind of soybean HRM-SNP molecular marker point marking method and its application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a soybean HRM-SNP molecular marker point marking method and application thereof.

Background technique

At present, the existing technologies commonly used in the industry are as follows:

So far, there are more than 20 methods for SNP typing detection. According to the need for gel electrophoresis and the degree of automation, SNP detection methods can be roughly divided into two categories. One is the SNP detection method based on gel electrophoresis, such as single-strand conformational polymorphism (Single-Strand Conformational Polymorphism, SSCP), denaturing gradient gel electrophoresis (Denatured Gradient Gel Electrophoresis, DGGE), cleavage amplification polymorphic sequence analysis (Cleaved Amplified Polymorphic Sequences, CAPS), allele-specific PCR (Allele Specific PCR, AS-PCR), etc.; It is a high-throughput, highly automated SNP detection method, such as direct sequencing, DNA chip, denaturing high performance liquid chromatography (Denaturing High Performance Liquid Chromatography, DHPLC), mass spectrometry detection, high-resolution melting curve (High-resolution Melting , HRM) etc.

The above SNP detection methods have their own advantages and disadvantages. Some can accurately detect SNP sites, such as DNA sequencing and DNA chip technology, but they are expensive and cannot be widely used; some operations are complicated and time-consuming, such as AS-PCR technology. ; Some have high throughput, low cost, and accurate results, but have high requirements for equipment hardware, such as HRM technology. An ideal SNP detection method should have the following characteristics: high sensitivity and accuracy; fast, simple, high-throughput; relatively low cost. But so far there is no detection method that fully meets the above characteristics, so a more suitable method can be selected according to the actual situation.

Soybean is the main oilseed and economic crop in my country, with a planting history of more than 5,000 years. Because of its rich nutrition, good flavor, high economic benefits, and diversified forms of harvesting, processing, and sales, it is popular in China, Japan and other Asian countries.

Although soybean originated in my country, it developed relatively late. Early breeding was mainly based on screening or cross-breeding of excellent varieties introduced from Taiwan Province or Japan, resulting in a narrow genetic basis for breeding resources, which is not conducive to the development of new varieties. At present, the Simple Sequence Repeat (SSR) markers, which are mainly used in various researches on soybeans, also have disadvantages such as relatively small number, complex analysis operations, and difficulty in determining the size of the markers so that they cannot be used across studies.

High-resolution melting curve (high-resolution melting, HRM) technology is a new type of SNP detection technology. In recent years, with the continuous development and improvement of this technology, it has been successfully applied to genotyping, mutation scanning, methylation research, etc., and has been successfully applied in the research of potato, alfalfa, citrus and other crops. Therefore, the development of new SNP molecular markers with higher density, easier analysis, and genotype determination that can be used across research has very important application value.

In summary, the problems in the prior art are:

(1) Simple Sequence Repeat (SSR) markers, which are currently mainly used in various researches on soybeans, also have disadvantages such as relatively small number, complex analysis operations, and difficulty in determining the size of the markers so that they cannot be used across studies.

(2) Due to the extremely complex soybean genome, HRM technology itself has relatively high requirements for primer design. During the primer design process, it is often encountered that the SNP site sequence is not unique in the genome, or the designed primers have non-unique The case of specific amplification.

The significance of solving the above technical problems:

Compared with the previous two generations of DNA molecular markers, SNP markers have high genetic stability, high density, rapid detection, and easy automatic analysis. SNPs have a high distribution frequency in the soybean genome, with an average of 273 bp There is one SNP, which is an important means for encrypting soybean genetic map, mapping QTL for important traits, and molecular marker-assisted selection.

Using Primer-BLAST for primer design can ensure the specificity of amplification and the high success rate of primer design. Since a large number of SNP sites were discarded and replaced during the preliminary primer design process, it is roughly estimated that the final success rate of primer design is higher than 85%.

These 101 SNP markers are evenly distributed on the 20 chromosomes of soybean. These markers can be used directly in the research, and the marker density can also be improved by using the above-mentioned method of the present invention to continuously develop new markers between two adjacent SNP sites. It can be used as a reference for other crops with known genome information.

Contents of the invention

Aiming at the problems in the prior art, the present invention provides a soybean HRM-SNP molecular marker method and its application.

The present invention is achieved in this way, a method for marking soybean HRM-SNP molecular markers, comprising:

Step 1: Development of soybean SNP markers: use the Genome browser tool in the database to select one SNP marker per 10Mb from the Williams 82 physical map, select 4 to 6 SNPs for each chromosome, and distribute them evenly on the 20 chromosomes of the soybean genome , to obtain the detailed information of each SNP marker;

Step 2: Genomic DNA Extraction of Soybean (Zhexian No. 9): Genomic DNA of the young leaves of Zhexian No. 9 was extracted according to the CTAB method, diluted to 25ng·μL ^-1 , and stored in a -20°C refrigerator for subsequent HRM analysis use;

The third step: high-resolution melting curve process and analysis: perform PCR amplification and HRM analysis on a quantitative PCR instrument; high-resolution melting curve analysis uses 480 software Gene Scanning module to obtain a better standardized melting curve;

Step 4: SNP typing of soybean (Zhexian No. 9) based on HRM technology: DNA of Zhexian No. 9 was amplified and analyzed by HRM in a quantitative PCR instrument;

Step 5: HRM typing verification of Soybean (Zhexian No. 9): Randomly perform Sanger sequencing on some SNP-marked PCR products to verify the accuracy of HRM analysis;

Step 6: Construction of soybean (Zhexian No. 9) SNP fingerprint:

Step 6: Construction of the SNP fingerprint of soybean (Zhexian No. 9): Using "Williams 82" as a control, use the developed 101 SNP markers to perform HRM typing on vegetable soybean "Zhexian No. Type results are listed in ascending order of chromosome number-physical map distance. For example, the typing result from the first marker on chromosome 1 to the last marker on chromosome 20 is: "AGTTCC...GGA", then this string of letters is the DNA fingerprint of "Zhexian No. 9". Therefore, the SNP map of Zhexian No. 9 is TCAACT AGCCA CTGCT CCTAG ATGAC CGCGA TGGGG TGGGC CACCA TCACTTCTT CTGGT TACCA TACCG TGGCG AGGTC TAAAC GATCTC CTCTG CTACT.

Further, in the extraction of soybean (Zhexian No. 9) genomic DNA, the genomic DNA of the young leaves of Zhexian No. 9 was extracted according to the improved CTAB method, diluted to 25ng·μL ^-1 , and stored in a -20°C refrigerator for subsequent HRM Analysis and use, the specific steps include:

(1) Extraction buffer 2% CTAB, 100mmol L ^-1 Tris-HCL, pH8.0; 20mmol L ^-1 EDTA; 2% PVP; 1.4mol L ^-1 Nacl, add 0.2% mercaptoethanol before use , preheated to 65°C;

(2) Take 0.05g of fresh leaves, cut them into pieces properly, put them into a 2.0ml centrifuge tube, add 1 steel ball with a diameter of 5mm, and 2-3 small steel balls with a diameter of 2mm;

(3) Add 1.0 mL of preheated extraction buffer into the centrifuge tube containing the sample and steel balls, cover the centrifuge tube cap, put it into the adapter and grind it in the TissueLyser-192 sample grinder for 60 seconds;

(4) Take out the centrifuge tube and place in a water bath at 65°C for 10-30min. Then take out the centrifuge tube and centrifuge at 1200rpm for 5-10min;

(5) Take 1 mL of the supernatant into a new 2 mL centrifuge tube, add 700 μL of phenol: chloroform: isoamyl alcohol = 25: 24: 1 by volume, slowly invert and mix several times; room temperature, centrifuge at 1200 rpm for 5-10 min;

(6) Take 800 μL supernatant to a new 2 mL centrifuge tube, add an equal volume of chloroform:isoamyl alcohol=24:1, slowly invert and mix several times; room temperature, centrifuge at 1200 rpm for 5-10 min;

(7) Transfer 600 μL of supernatant to a new 1.5 mL centrifuge tube; add 400 μL of pre-cooled isopropanol, slowly invert and mix, and place in a -20°C refrigerator for 30 minutes;

(8) Centrifuge at 1200rpm for 10min at room temperature, discard the supernatant;

(9) Add 1 mL of pre-cooled 70% ethanol to wash the DNA, centrifuge at 1200 rpm for 10 min, and discard the supernatant;

(10) Put the DNA into an ultra-clean workbench to dry, and add 100-200 μL of deionized water to dissolve the DNA;

(11) Add 21μL 0mg/mL RNase and bathe in 37℃ water for 30min;

(12) 5 μL of DNA was taken, and the quality of the DNA was detected by 1% agarose gel electrophoresis, and the concentration and quality of the DNA were detected by a UVS-99 trace nucleic acid detector.

Further, in the third step, the high-resolution melting curve process and analysis,

The combination of 20 μL reaction system is: 2 μL DNA 50ng, 4 μL 5x EVAGreen Realtime PCR Mix, 0.4 μL each of 10 μmol L ^-1 upstream and downstream primers, and the rest is made up with water; the mixed system is placed in a 96-well PCR plate, and the sealing film is attached Instantaneous centrifugation, placed in the instrument for reaction;

The reaction program is: pre-denaturation at 95°C for 15 minutes, after 45 regular cycles, followed by high-resolution melting, and the final product is cooled to 40°C for 10s;

The cycle program is 95°C for 15s, 60°C for 20s, 72°C for 20s, a total of 45 cycles;

The high-resolution melting process is: 1 min at 95 °C, 1 min at 40 °C, 1 s at 65 °C, read the melting curve at 65-95 °C, the temperature resolution is 0.02 °C, and the fluorescence information is collected continuously at a rate of 25 times per °C.

Further, in the fourth step, the HRM-based SNP typing results of soybean (Zhexian No. ) to represent.

Another object of the present invention is to provide a soybean variety DNA fingerprint library constructed by using the soybean HRM-SNP molecular marker method.

In summary, the advantages and positive effects of the present invention are:

The invention has high throughput and short analysis time: the analysis of 96 or 384 samples can be completed at one time, and is suitable for large-scale development and detection of SNP sites.

The invention is easy to operate: no sequence-specific probes and subsequent sequencing are required, only PCR primers need to be designed, PCR reaction is performed, and then HRM is directly performed to complete the genotype analysis of the sample.

The invention has good specificity: the PCR product does not need subsequent treatment, and the tube is completely closed.

The present invention has good generalizability: the 101 SNP markers developed by the present invention can be directly used in soybean fingerprint construction, germplasm identification, variety protection, genetic diversity analysis and other researches; it can also be used in other crops with known genome sequences Similar markup development for .

Description of drawings

Fig. 1 is the SNP marker developed based on the HRM technology and its distribution on the chromosome provided by the embodiment of the present invention.

In the figure: the left side is the name of dbSNP; the right side is the position (bp) of the SNP on the chromosome.

Fig. 2 is a diagram of two cases of SNP typing based on HRM technology provided by the embodiment of the present invention.

In the figure: A-D: the typing results of the non-polymorphic marker ss715590226 (Williams 82 and Zhexian No. 9); E-H: the typing results of the polymorphic marker ss71562667;

A, E: melting peak; B, F: normalized melting curve; C, G: normalized temperature difference view; D, H: Williams 82 and Zhexian No. 9. Three biological replicates were performed for each species.

Fig. 3 is a schematic diagram of a part of the samples of the normalized melting curve and PCR product sequencing provided by the embodiment of the present invention.

In the figure: the SNP typing of Zhexian No. 9 and Williams 82 were sequenced and marked near the corresponding normalized melting curves, A-F: the SNP typing results of ss715626377, ss715627029, ss715585535, ss715599654, ss715612033, ss715624203 respectively.

Fig. 4 is the fingerprint of Zhexian No. 9 constructed based on HRM-based SNP markers provided by the embodiment of the present invention.

In the figure: The genotype of each SNP locus is sorted by its chromosome and physical location. The polymorphic SNP sites of Williams 82 and Zhexian 9 are marked in red.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

Currently, Simple Sequence Repeat (SSR) markers, which are mainly used in various researches on soybeans, also have disadvantages such as relatively small number, complex analysis operations, and difficulty in determining the size of the markers so that they cannot be used across research.

According to the complete gene sequence information of soybean variety "Williams 82", the present invention develops SNP molecular markers based on HRM technology that are evenly distributed on 20 soybean chromosomes, and applies the developed markers to soybean variety "Zhexian No. 9" (Zhejiang Province Crop and nuclear technology of the Academy of Agricultural Sciences uses the genotyping and DNA fingerprinting of the fresh soybean variety bred by the Institute, National Examination Dou 2009023), to verify the reliability of the developed SNP marker method and the availability of the developed SNP marker. (Zhexian No. 9 is a good variety in the prior art, and the main innovation point of the present invention lies in this new molecular marking method.)

The soybean HRM-SNP molecular marker point marking method provided by the embodiment of the present invention includes:

Step 1: Development of soybean SNP markers:

According to the latest genome sequence information (Glyma.Wm82.a2, Gmax2.0) of the SoyBase and the Soybean Breeder'Toolbox database (https://www.soybase.org/), the physical map length of Williams 82 ranges from 34,766,867bp to 58,018,742bp , with an average length of 47,459,169 bp. Use the Genome browser tool in the database to select approximately one SNP marker per 10Mb from the "Williams 82" physical map, and select 4 to 6 SNPs for each chromosome, which are evenly distributed on the 20 chromosomes of the soybean genome (Table 1). Detailed information is available for each SNP marker, including dbSNP name, SNP position, SNP allele, and 121bp genomic sequence (Table 2). Use the Primer-Blast of the NCBI database (https://www.ncbi.nlm.nih.gov/tools/primer-blast), paste the 121bp genome sequence of each SNP into the PCR template box, set the relevant parameters, and design online the desired specific primers. The parameters for designing primers are as follows: forward primer range 1-60bp, reverse primer range 62-121bp, Tm value is between 59-63°C, the best is 61°C, PCR product length is 60-100 bp, database selection Refseq representative genomes, species selection Glycine max(L.) Merr.(taxid:3847).

Finally, a total of 101 SNP loci distributed evenly on 20 soybean chromosomes for HRM analysis were selected and successfully transformed into SNP markers. Among them, there are 6 SNP markers on Gm01 and Gm18, 4 on Gm11, and 5 on other chromosomes ( FIG. 1 ). The total average spacing of these 101 SNP markers on the chromosome is 9,397,855bp. The primers were synthesized by Hangzhou Qingke Zixi Biotechnology Co., Ltd. at a concentration of 10 μmol·L ^-1 , and stored at -20°C for future use.

Table 1 Genome information of "Williams 82" and the number of SNPs selected on each chromosome

Table 2 SNP sites and primer information based on HRM technology SNP analysis

The second step: "Zhexian No. 9" genomic DNA extraction

"Zhexian No. 9" was bred by the Institute of Crop and Nuclear Technology Utilization of Zhejiang Academy of Agricultural Sciences. It is a fresh spring soybean. This variety has good yield and good commerciality.

The genomic DNA of young leaves of "Zhexian No. 9" was extracted according to the CTAB method, diluted to 25 ng·μL ^-1 , and stored in a -20°C refrigerator for subsequent HRM analysis. The specific steps are as follows:

(1) Extraction buffer (2% CTAB, 100mmol L ^-1 Tris-HCL, pH8.0; 20mmol L ^-1 EDTA; 2% PVP; 1.4mol L ^-1 NaCl), add 0.2% of Mercaptoethanol, preheated to 65°C.

(2) Take 0.05g of fresh leaves (about 4cm2), cut them into pieces (about 3-4mm in size), put them into a 2.0ml centrifuge tube, add 1 steel ball with a diameter of 5mm, and 2-3 small steel balls with a diameter of 2mm. Pay attention to cleaning the scissors in time to avoid cross-contamination of samples.

(3) Add 1.0 mL of preheated extraction buffer into the centrifuge tube containing the sample and steel balls, cover the centrifuge tube cap, install the adapter and grind in a TissueLyser-192 sample mill for 60 seconds (frequency 70).

(4) Take out the centrifuge tube and place in a water bath at 65°C for 10-30min. Then take out the centrifuge tube and centrifuge at 1200rpm for 5-10min.

(5) Take 1 mL of the supernatant to a new 2 mL centrifuge tube, add 700 μL of phenol:chloroform:isoamyl alcohol (25:24:1), and mix by inverting slowly several times. Centrifuge at 1200rpm for 5-10min at room temperature.

(6) Take 800 μL of supernatant to a new 2 mL centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), and slowly invert and mix several times. Centrifuge at 1200rpm for 5-10min at room temperature. Be careful not to pipette into the middle layer of the liquid phase.

(7) Transfer 600 μL supernatant to a new 1.5 mL centrifuge tube. Add 400 μL of pre-cooled isopropanol (filamentous DNA can be seen at this time), mix slowly by inversion, and place in a -20°C refrigerator for 30 minutes. Be careful not to pipette into the middle layer of the liquid phase.

(8) Centrifuge at 1200 rpm for 10 min at room temperature and discard the supernatant.

(9) Add 1 mL of pre-cooled 70% ethanol to wash the DNA, centrifuge at 1200 rpm for 10 min, and discard the supernatant.

(10) Put the DNA into an ultra-clean workbench to dry, and add 100-200 μL of deionized water to dissolve the DNA.

(11) Add 21 μL of 0 mg/mL RNase and bathe in 37°C water for 30 minutes (the water bath step can be omitted).

(12) 5 μL of DNA was taken, and the quality of the DNA was detected by 1% agarose gel electrophoresis, and the concentration and quality of the DNA were detected by a UVS-99 trace nucleic acid detector (ACTGene, USA).

Step 3: High resolution melting curve process and analysis:

at Roche PCR amplification and HRM analysis were performed on a 480II quantitative PCR instrument (Roche). After preliminary experiments, a better reaction system was established. The composition of the 20 μL reaction system was: 2 μL DNA (50ng), 4 μL 5xEVA Green Realtime PCR Mix (Gene Solution), 0.4 μL (10 μmol·L ^-1 ) each of the upstream and downstream primers, and the rest was made up with water. The mixed system was placed in a 96-well PCR plate, sealed with a plate film, centrifuged for a short time, and placed in the instrument for reaction. The reaction program was: 95°C pre-denaturation for 15 min, 45 regular cycles, followed by high-resolution melting, and the final product was cooled to 40°C for 10 s. The cycle program is 95°C for 15s, 60°C for 20s, and 72°C for 20s, a total of 45 cycles. The high-resolution melting process is: 95°C for 1min, 40°C for 1min, 65°C for 1s, read the melting curve at 65-95°C, the temperature resolution is 0.02°C, and the fluorescence information is collected continuously at a rate of 25 times per degree Celsius.

High resolution melting curve analysis using 480 software (v1.5.0) Gene Scanning module. Adjust the "Pre-melt Slider Settings" and "Post-melt Slider Settings" values according to the actual situation to obtain better normalized melting curves (Normalized Melting Curves). The "Temperature Shift" threshold is generally set to 1, and in some cases it is set to 0 for better analysis results.

The fourth step: "Zhexian No. 9" SNP typing based on HRM technology:

"Zhexian No. 9" DNA in Roche Amplification and HRM analysis were performed in a 480II quantitative PCR instrument (Roche). Theoretically, there are 3 types of SNP typing based on HRM: allele I (same as the reference variety "Williams 82"), allele II (different from the reference variety "Williams 82"), and heterozygous ( The SNP site is in a heterozygous state). However, due to the high genome homozygosity of "Williams 82" and "Zhexian No. 9", the present invention only found two cases, and no heterozygosity occurred. The typing results can be represented by Melting Peaks, Normalized Melting Curves, and Normalized & Temp-Shifted Difference Plots.

Step 5: "Zhexian No. 9" HRM typing verification

In addition to genotype grouping and comparison of melting curves, some SNP-marked PCR products were randomly subjected to Sanger sequencing to verify the accuracy of the HRM analysis.

Step 6: Construction of the SNP fingerprint of "Zhexian No. 9":

After confirming the accuracy of this method by sequencing, "Zhexian No. 9" was genotyped using the developed 101 SNP markers based on HRM technology that are evenly distributed on soybean chromosomes. After analysis, it was found that among these markers, 34 (33.7%) SNP sites were polymorphic between "Zhexian No. 9" and "Williams 82" (Fig. 4). The typing results are listed in ascending order of chromosome number-physical map distance, which is the SNP fingerprint of Zhexian No. 9.

The present invention will be further described below in conjunction with specific experiments.

Construction of SNP Fingerprint of Vegetable Soybean "Zhexian No. 9"

(1) SNP typing of vegetable soybean "Zhexian No. 9" based on HRM technology

Figure 2 lists two examples of SNP typing of "Zhexian No. 9" based on HRM technology: the typing results of ss715590226 and ss715626667 markers.

The SNP typing results of "Zhexian No. 9" at the ss715590226 site (G/A) showed that there was no significant difference in the melting peak (Figure 2A) and the normalized melting curve (Figure 2B), and the normalized temperature difference view (Figure 2C) There is also no regular grouping, indicating that there is no difference in the analysis site of "Zhexian No. 9". It is known that the genotype of the reference variety "Williams 82" at this locus is G, and it can be inferred that the genotype of "Zhexian No. 9" is the same as that of the reference variety.

The SNP typing results of "Zhexian No. 9" at the ss715626667 site (C/A) showed that the melting peak (Fig. 2E), the normalized melting curve (Fig. 2F) and the normalized temperature difference view (Fig. 3) all appeared obvious Two sets of specific curves indicated that there were differences at this locus among the analyzed germplasm. It is known that the genotype of the reference variety "Williams 82" at this locus is C. It can be known that "Zhexian No. 9" and "Williams 82" have different curves, and it can be inferred that the genotype of "Zhexian No. 9" is A.

(2) HRM typing verification of vegetable soybean "Zhexian No. 9"

Concordance between HRM-based SNP genotyping and Sanger sequencing was observed on all these sequenced SNP loci, indicating that the HRM-based SNP analysis system established in this study is accurate and can be used for soybean SNP genes Typing.

Figure 3 shows a partial HRM genotyped and PCR-sequenced sample.

(3) Construction of SNP fingerprint of vegetable soybean "Zhexian No. 9"

After confirming the accuracy of this method by sequencing, the vegetable soybean variety "Zhexian No. 9" was genotyped using the developed 101 SNP markers based on HRM technology that were evenly distributed on the soybean chromosome. After analysis, it was found that among these markers, 34 (33.7%) SNP sites were polymorphic between "Zhexian No. 9" and "Williams 82" (Fig. 4). The typing results are listed in ascending order of chromosome number-physical map distance, so the SNP map of Zhexian No. 9 is SEQ ID NO: 1: "TCAACT AGCCA CTGCT CCTAG ATGAC CGCGA TGGGG TGGGC CACCATCACT TCTT CTGGT TACCA TACCG TGGCG AGGTC TAAAC GATCTC CTCTG CTACT".

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

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Claims (5)

1. a method for marking soybean HRM-SNP molecular markers, characterized in that, the method for marking soybean HRM-SNP molecular markers comprises: Development of soybean SNP markers: use the Genome browser tool in the database to select one SNP marker per 10Mb from the Williams 82 physical map, select 4 to 6 SNPs for each chromosome, and distribute them evenly on the 20 chromosomes of the soybean genome. Details of SNP markers; Soybean genomic DNA extraction: Genomic DNA of young soybean leaves was extracted according to the improved CTAB method, diluted to 25ng·μL ^-1 , and stored in a -20°C refrigerator for subsequent HRM analysis; High-resolution melting curve process and analysis: PCR amplification and HRM analysis are performed on a quantitative PCR instrument; high-resolution melting curve analysis uses 480 software Gene Scanning module to obtain a better standardized melting curve; Soybean SNP typing based on HRM technology: Soybean DNA is amplified and analyzed by HRM in a quantitative PCR instrument; Soybean HRM typing verification: Randomly perform Sanger sequencing on some SNP-marked PCR products to verify the accuracy of HRM analysis; Construction of soybean SNP fingerprints: Using Williams 82 as a control, the 101 SNP markers developed were used for HRM typing of vegetable soybeans, and the typing results were listed in order of chromosome number-physical map distance from small to large. 2. soybean HRM-SNP molecular marker point marking method as claimed in claim 1, is characterized in that, In the extraction of soybean genomic DNA, the soybean was Zhexian No. 9. The genomic DNA of the young leaves of Zhexian No. 9 was extracted according to the improved CTAB method, diluted to 25ng·μL ^-1 , and stored in a -20°C refrigerator for subsequent HRM analysis Use, the specific steps include: (1) Extraction buffer 2% CTAB, 100mmol L ^-1 Tris-HCL, pH8.0; 20mmol L ^-1 EDTA; 2% PVP; 1.4mol L ^-1 Nacl, add 0.2% mercaptoethanol before use , preheated to 65°C; (2) Take 0.05g of fresh leaves, cut them into pieces properly, put them into a 2.0ml centrifuge tube, add 1 steel ball with a diameter of 5mm, and 2-3 small steel balls with a diameter of 2mm; (3) Add 1.0 mL of preheated extraction buffer into the centrifuge tube containing the sample and steel balls, cover the centrifuge tube cap, put it into the adapter and grind it in the TissueLyser-192 sample grinder for 60 seconds; (4) Take out the centrifuge tube and place in a water bath at 65°C for 10-30min. Then take out the centrifuge tube and centrifuge at 1200rpm for 5-10min; (5) Take 1 mL of the supernatant into a new 2 mL centrifuge tube, add 700 μL of phenol: chloroform: isoamyl alcohol = 25: 24: 1 by volume, and slowly invert and mix several times; room temperature, centrifuge at 1200 rpm for 5-10 min; (6) Take 800 μL supernatant to a new 2 mL centrifuge tube, add an equal volume of chloroform:isoamyl alcohol=24:1, slowly invert and mix several times; room temperature, centrifuge at 1200 rpm for 5-10 min; (7) Transfer 600 μL of supernatant to a new 1.5 mL centrifuge tube; add 400 μL of pre-cooled isopropanol, slowly invert and mix, and place in a -20°C refrigerator for 30 minutes; (8) Centrifuge at 1200rpm for 10min at room temperature, discard the supernatant; (9) Add 1 mL of pre-cooled 70% ethanol to wash the DNA, centrifuge at 1200 rpm for 10 min, and discard the supernatant; (10) Put the DNA into an ultra-clean workbench to dry, and add 100-200 μL of deionized water to dissolve the DNA; (11) Add 21μL 0mg/mL RNase and bathe in 37℃ water for 30min; (12) Take 5 μL of DNA, and use 1% agarose gel electrophoresis to detect the quality of the DNA, and a UVS-99 trace nucleic acid detector to detect the DNA concentration and quality. 3. soybean HRM-SNP molecular marker point marking method as claimed in claim 1, is characterized in that, In high-resolution melting curve processing and analysis, The combination of the 20 μL reaction system is: 2 μL DNA 50ng, 4 μL 5x EVAGreen Realtime PCR Mix, 0.4 μL each of the upstream and downstream primers of 10 μmol L ^-1 , and make up the rest with water; the mixed system is placed in a 96-well PCR plate, and the sealing film is attached Instantaneous centrifugation, placed in the instrument for reaction; The reaction program is: pre-denaturation at 95°C for 15 minutes, after 45 regular cycles, followed by high-resolution melting, and the final product is cooled to 40°C for 10s; The cycle program is 95°C for 15s, 60°C for 20s, 72°C for 20s, a total of 45 cycles; The high-resolution melting process is: 1 min at 95 °C, 1 min at 40 °C, 1 s at 65 °C, read the melting curve at 65-95 °C, the temperature resolution is 0.02 °C, and the fluorescence information is collected continuously at a rate of 25 times per °C. 4. soybean HRM-SNP molecular marker point marking method as claimed in claim 1, is characterized in that, The SNP typing results of soybean based on HRM technology are represented by Melting Peaks, Normalized Melting Curves, and Normalized&Temp-Shifted Difference Plots. 5. A DNA fingerprint library of soybean varieties constructed by utilizing the soybean HRM-SNP molecular marker method according to claim 1.