A method for preparing grape variety DNA molecular identity card using SSR fingerprint and its application

Abstract

The present invention relates to a method for preparing a grape variety DNA molecular ID card using SSR fingerprint and its application; the present invention randomly develops a certain number of SSR marker primers, and further screens out primers with clear fingerprints and high polymorphism for amplifying the fingerprint spectrum of the grape variety, and then develops a DNA molecular ID card of the grape variety that can meet the application of accurate identification based on whether the spectrum has 0-1 matrix information. The present invention also applies Excel and Heatmap to the technology of rapid and accurate variety identification, and greatly improves the efficiency of DNA fingerprint conversion through DNA fingerprint data analysis, comparison and rapid retrieval, and has the characteristics of low cost, fast operation, simplicity, high efficiency, accuracy and, which can not only overcome the shortcomings of traditional morphological identification measures for accurate identification of a large number of varieties, but also is convenient for rapid and efficient identification of grape varieties and protection of new variety property rights, and can greatly improve the efficiency of preservation and management of germplasm resources.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology, and more particularly to a method for producing a DNA molecular identity card using SSR fingerprints and an application thereof, so as to achieve the effect of efficient, rapid and accurate identification of different grape varieties. Background Art

[0002] Grape is one of the important economic crops in my country, with a large planting area, high nutritional value and extremely rich germplasm resources. With the continuous increase in the selection and promotion of excellent varieties, the speed of variety approval has accelerated, and the number of new varieties has increased year by year, the protection of variety intellectual property rights has become a top priority. Due to the frequent exchange of genes between varieties, the number of varieties is increasing. In production, there are often phenomena such as homonymous and fake and real. Accurate identification is an important basis for the preservation, research, development and utilization of grape germplasm resources and the protection of new variety rights, and it is also an important guarantee to ensure the safety and reliability of introduced varieties.

[0003] Therefore, it is of great significance to improve the efficiency of DNA fingerprint conversion, develop a grape variety identification method with high accuracy, low cost, simple operation and high efficiency, and use it to produce a DNA molecular identity card for grape variety resources.

[0004] Molecular markers that can reveal polymorphism at the DNA level have been widely used in fruit tree gene positioning and cloning, kinship analysis or variety identification, evolutionary research, etc. Compared with other DNA markers, SSR markers have richness, high polymorphism, codominant inheritance and high repeatability. SSR markers have the advantages of good stability, simple and easy operation, abundant number of SSR loci in the whole grape genome, and high diversity. With the rapid development of sequencing technology, a large number of SSR loci can be identified quickly and cost-effectively through next-generation sequencing (NGS) technology. There are about 286192~627429 SSR loci on the grape genome, and there are about 1 SSR loci per kb. The results of kinship analysis of different grape varieties are similar to SNP. The SSR loci mining based on the whole genome is efficient, the number of output SSR markers is higher, and it has better genome coverage. It is currently the preferred marker technology. With the development and improvement of DNA fingerprint identification technology, SSR fingerprints are digitized. At present, there is no method to directly use DNA fingerprints to construct molecular identity cards.

[0005] Heatmap is a type of chart used to visualize data. It uses color coding and spatial coordinates to show the density and distribution of data points. It can intuitively display the regions and trends in a large number of data sets, and is used to discover patterns and regularities in the data. Heatmaps are used to represent two-dimensional arrays (matrices). The principle of hierarchical clustering is to classify the rows or columns of the matrix according to the similarity of data points, and then obtain the correlation in the data. From the perspective of samples, cluster analysis can observe whether different samples collected are of the same category or the same sample. Heatmaps are used to analyze, compare and quickly retrieve DNA fingerprints (variety ID cards) of different varieties. Its visualization of the differences in digital DNA fingerprints effectively improves the efficiency of DNA fingerprint conversion, making it possible to use Heatmap for germplasm variety identification.

[0006] Therefore, based on the above principles, the development of an economical, efficient, accurate and stable variety identification method for rapid and accurate identification of grape varieties can provide theoretical support for the analysis of grape variety identification, genetic evolution, kinship, taxonomic status, etc., which is of great significance for improving the management efficiency of my country's grape germplasm resources and the ability to protect varieties. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for making a DNA molecular ID card of a grape variety using SSR fingerprints and its application. The present invention uses 50 grape varieties as the objects for making DNA molecular ID cards and rapid identification, provides a set of SSR molecular marker combinations for identifying the grape germplasm resource library, quickly converts DNA fingerprints into a 0-1 matrix, and uses the Excel-concat formula to merge them into a molecular ID number composed of 0 and 1. The method has concise coding and can provide a simple tool for variety property protection.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] In the first aspect, the present invention protects a method for making a grape variety DNA molecular ID card using SSR fingerprints, using Excel tools to directly generate a variety molecular ID number from the 0-1 matrix information converted from the DNA fingerprint, as follows:

[0010] (1) Using the MISA tool, SSR primer sequences were randomly designed and developed based on the whole grape genome;

[0011] (2) Collecting young leaves of grape variety resources as plant materials and extracting sample genomic DNA;

[0012] (3) Randomly select 8-10 sample DNAs as amplification templates, use the SSR primers in step (1) for PCR amplification, and preliminarily screen the primers to select primers with clear bands and polymorphism among varieties. Each band of each pair of primers is used as a specific site. The sequence of the sorted primers is shown in SEQ ID No: 1-20;

[0013] (4) Use the selected primer combinations to perform PCR detection on grape variety resource samples in sequence;

[0014] (5) Detect the PCR amplification results by polyacrylamide gel electrophoresis to obtain the DNA fingerprint of grape variety resources;

[0015] (6) Convert the polymorphic bands in the DNA fingerprint of the variety resources into a 0-1 matrix: the rows and columns are different samples, and the vertical columns are the sorted primer sites. Count the target band information amplified from different varieties at different sites, where the presence of a band is recorded as "1" and the absence of a band is recorded as "0";

[0016] (7) Use the Excel-concat data merging formula to connect the constructed 0-1 matrix rows and columns to obtain the molecular ID number of the corresponding variety resource.

[0017] In a specific embodiment, the grape variety is selected from one or more of Aligot, Armas, Aibel, Elwein, Iona, Ayun Queen, Pinot Blanc, White Collette, White Banana, White Xiexi, White Feather, Baichel, Beisaihun, Bud Rose, Green Pearl, Pollux, Bugisuri, Bresa, Cabernet Sauvignon, Crisp Peak, Crisp Red, Dabao, Dilawa, Emerald Rose, Fengbao, Fenghou, Fengshou, Fusling, Takasago, Guifei Rose, Guirenxiang, Hamilak, Black Olin, Kuroshio, Black Peak, Black Rose, Black Queen, Black Saibil, Black Vanisico, Red Igawa, Red Shuangwei, Red Banana, Red Yi, Pinot Gris, Venus Seedless, Igawa, Wine White, Kyoho, Giant Whale and Giant Rose.

[0018] The present invention also protects the grape variety DNA molecular identity card prepared by the aforementioned method of preparing the grape variety DNA molecular identity card using SSR fingerprint.

[0019] The grape variety molecular ID card composed of 0 and 1 constructed by the present invention can simply and quickly distinguish the characteristics of different varieties, has the advantages of high efficiency, accuracy and convenience, and is easy to record, analyze, compare and retrieve; not only is the encoding concise, but it also makes the distinction, identification and comparison of varieties more convenient and intuitive.

[0020] In a specific implementation scheme, in step (2), the genomic DNA extraction method is the CTAB method: the young grape leaves are placed in liquid nitrogen and ground completely, and then the genomic DNA is extracted by the CTAB method and diluted to a concentration of 20 ng / μL.

[0021] In a specific embodiment, in step (3), the PCR amplification system is a system that includes 2 μL of ddH2O, 5 μL of 2×Taq master mix, 1 μL of 20 ng / μL template DNA, 1 μL of 10 μM forward primer and 1 μL of 10 μM reverse primer, based on 10 μL.

[0022] In a specific embodiment, the PCR amplification program is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55-60°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 10 min.

[0023] In a specific embodiment, in step (5), 8% polyacrylamide gel is used for gel electrophoresis, and the 8% gel preparation includes: 30 mL of ddH2O, 15 mL of 30% gel making solution, 5 mL of 10×TBE, 80 μL of 10% AP3, and 40 μL of TEMED; the gel electrophoresis program is: voltage 200 V, current 400 mA, and time 1.3 h; and the gel color is developed using silver staining.

[0024] In a second aspect, the present invention provides a primer combination, wherein the primer combination consists of 10 pairs of primers, and the primer sequences are shown in SEQ ID Nos: 1 to 20.

[0025] In a third aspect, the present invention also protects a reagent containing the primer combination described above.

[0026] In a fourth aspect, the present invention also protects a kit containing the primer combination or the reagent described above.

[0027] In a fifth aspect, the present invention also protects the use of the method for preparing a grape variety DNA molecular identity card using SSR fingerprints, the primer combination, the reagent, or the kit described above in any of the following (A1) to (A4):

[0028] (A1) Application in identification of unknown grape varieties;

[0029] (A2) Use in the preparation of products for identification of unknown grape varieties;

[0030] (A3) Application in distinguishing confusing grape varieties;

[0031] (A4) Use in the preparation of products for differentiating grape varieties;

[0032] The grape variety is selected from one or more of Aligot, Armas, Aibel, Elwein, Iona, Ayun Queen, Pinot Blanc, White Collette, White Banana, White Sheshi, White Feather, Baichel, Beisaihun, Bud Rose, Green Pearl, Pollux, Bugisuri, Bresa, Cabernet Sauvignon, Crisp Peak, Crisp Red, Dabao, Dilawa, Emerald Rose, Fengbao, Fenghou, Fengshou, Fusling, Takasago, Guifei Rose, Guirenxiang, Hamilak, Black Olin, Kuroshio, Black Peak, Black Rose, Black Queen, Black Sebil, Black Vanisico, Red Igawa, Red Shuangwei, Red Banana, Red Yi, Pinot Gris, Venus Seedless, Igawa, Wine White, Kyoho, Giant Whale and Giant Rose.

[0033] In a sixth aspect, the present invention protects a method for identifying unknown grape varieties, which imports the 0-1 matrix statistically generated in Excel into TBtools in the form of a two-dimensional array, thereby constructing a HeatMap hierarchical clustering heat map for visual variety identification, as follows:

[0034] (1) Use the CTAB method to extract the genomic DNA of the sample to be tested;

[0035] (2) using the primer sequences shown in SEQ ID Nos: 1 to 20 to perform PCR amplification on the genomic DNA of the unknown sample;

[0036] (3) The PCR amplification product was detected by 8% polyacrylamide gel electrophoresis (PAGE), and the electrophoresis results were counted. The 0-1 matrix information of the unknown samples to be detected was added to the data table in sequence according to the 0-1 matrix construction method mentioned above;

[0037] (4) Use TBtools software, select the HeatMap heat map tool in the HeatMap Illustrator module in Graphics, input the two-dimensional array format information of the 0-1 matrix constructed in step (3), click start, get preliminary results, select Control Dialog for heat map analysis, select Euclidean as Dist Method to calculate the genetic distance between samples, select Complete as Cluster Method to perform hierarchical clustering analysis, and the cluster heat map is presented, realizing hierarchical clustering with the molecular ID number of each sample as the data point;

[0038] (5) Analyze the results of the HeatMap clustering heat map: If the unknown sample to be tested is clustered with one of the samples in the variety resource library and the DNA fingerprint information is completely matched, the real variety name of the unknown sample is the corresponding variety of the sample;

[0039] The grape variety is selected from one or more of Aligot, Armas, Aibel, Elwein, Iona, Ayun Queen, Pinot Blanc, White Collette, White Banana, White Sheshi, White Feather, Baichel, Beisaihun, Bud Rose, Green Pearl, Pollux, Bugisuri, Bresa, Cabernet Sauvignon, Crisp Peak, Crisp Red, Dabao, Dilawa, Emerald Rose, Fengbao, Fenghou, Fengshou, Fusling, Takasago, Guifei Rose, Guirenxiang, Hamilak, Black Olin, Kuroshio, Black Peak, Black Rose, Black Queen, Black Sebil, Black Vanisico, Red Igawa, Red Shuangwei, Red Banana, Red Yi, Pinot Gris, Venus Seedless, Igawa, Wine White, Kyoho, Giant Whale and Giant Rose.

[0040] In the seventh aspect, the present invention protects a method for distinguishing confused grape varieties, in which Excel directly compares the DNA fingerprint information of known varieties and confused varieties and combines the HeatMap hierarchical clustering heat map to perform visual variety identification, as follows:

[0041] (1) Use the CTAB method to extract the genomic DNA of the sample to be tested;

[0042] (2) using the primer sequences shown in SEQ ID Nos: 1 to 20 to perform PCR amplification on the genomic DNA of the confounding sample;

[0043] (3) The PCR amplification products were detected by 8% polyacrylamide gel electrophoresis (PAGE), and the electrophoresis results were statistically analyzed to construct a 0-1 matrix of the samples to be tested. The DNA fingerprint information of known species was added to this matrix to form a new 0-1 matrix;

[0044] (4) Using the Excel conditional format, set a two-color scale staining rule for the 0-1 matrix containing the samples to be tested constructed in step (3), setting the value 1 and the value 0 to different colors, and visually distinguishing the differences in the sample DNA fingerprints. If two or more colors in the confused varieties are exactly the same, the confused varieties are the same variety;

[0045] (5) Use Excel-concat to develop its molecular ID number and perform differential sample comparison to achieve auxiliary differentiation: if the ID number of the tested variety is exactly the same as that of the known variety, then the tested sample corresponds to the known variety;

[0046] The grape variety is selected from one or more of Aligot, Armas, Aibel, Elwein, Iona, Ayun Queen, Pinot Blanc, White Collette, White Banana, White Sheshi, White Feather, Baichel, Beisaihun, Bud Rose, Green Pearl, Pollux, Bugisuri, Bresa, Cabernet Sauvignon, Crisp Peak, Crisp Red, Dabao, Dilawa, Emerald Rose, Fengbao, Fenghou, Fengshou, Fusling, Takasago, Guifei Rose, Guirenxiang, Hamilak, Black Olin, Kuroshio, Black Peak, Black Rose, Black Queen, Black Sebil, Black Vanisico, Red Igawa, Red Shuangwei, Red Banana, Red Yi, Pinot Gris, Venus Seedless, Igawa, Wine White, Kyoho, Giant Whale and Giant Rose.

[0047] The present invention uses the HeatMap hierarchical clustering method to visualize the differences of grape variety DNA molecular ID fingerprints obtained by developing SSR molecular markers based on the whole genome, and uses the hierarchical clustering algorithm tool to quickly match unknown samples to be tested / confused samples with variety resource library samples, which can solve the problem of low efficiency in distinguishing varieties in the germplasm resource library using identity card numbers with too many character string encoding digits. The method is simple and has higher efficiency and accuracy.

[0048] The present invention has the following technical effects compared with the prior art:

[0049] (1) The present invention screens specific SSR marker combinations for different grape varieties to identify the varieties and quickly construct molecular ID numbers, which can be used for variety intellectual property protection.

[0050] (2) The present invention combines the variety ID construction method with the HeatMap hierarchical clustering method, which can be used for the visual rapid identification of unknown / confusing grape varieties. This method greatly improves the efficiency of DNA fingerprint conversion through data analysis, comparison and rapid retrieval, has high accuracy and stability of discrimination results, is simple to operate, and takes a short time to identify. It overcomes the defects of cumbersome variety identification technology and is easy to promote and apply in practice.

[0051] (3) The principles and technical processes of the present invention are also suitable for the construction and precise identification of DNA molecular identity cards of other fruit trees and tea tree variety resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a schematic diagram of the construction and application process of the grape germplasm resource molecular identity card provided by the present invention.

[0053] Figure 2 This is the PAGE electrophoresis run of 50 grape samples detected under a specific SSR marker combination.

[0054] Figure 3 This is the PAGE electrophoresis run of 5 randomly selected grape samples tested under a specific SSR marker combination.

[0055] Figure 4 This is a visual identification result diagram of 5 unknown grape samples.

[0056] Figure 5 This is a visual identification result diagram of 5 confused grape samples.

[0057] Figure 6 This is the DNA fingerprint 0-1 matrix of the sample to be tested in Example 3. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0059] Unless otherwise specified, the samples, consumables and reagents listed or used in the context of the present invention can be obtained commercially. The experimental methods used in the present invention, such as DNA extraction, PCR, polyacrylamide gel electrophoresis, etc., are all conventional methods and techniques in the art.

[0060] Example 1 Construction of Grape Variety Molecular ID Card

[0061] 1. Test material selection

[0062] The experimental materials are 50 grape germplasm resources provided by the Zhengzhou Fruit Research Institute of the Chinese Academy of Agricultural Sciences. All samples were picked on June 15, 2023.

[0063] Table 1: Information table of 50 grape samples for trial supply

[0064]

[0065] 2. Sample preparation and DNA extraction

[0066] Collect fresh leaves of grape varieties (Table 1) and extract grape genomic DNA using the CTAB method:

[0067] (1) Take 100 mg of leaf tissue in a grinding tube, add liquid nitrogen and quickly grind into powder;

[0068] (2) Add 1 mL of 65°C preheated CTAB extraction solution and 1 μL of 20 mg / mL RNase A, mix well with a vortex mixer, and incubate at 65°C for 60 min.

[0069] (3) After cooling to room temperature, centrifuge at 10,000 rpm for 5 min, aspirate the supernatant, transfer to a 1.5 ml centrifuge tube, add an equal volume of chloroform and isoamyl alcohol (24:1), and centrifuge at 10,000 rpm for 20 min;

[0070] (4) Take the supernatant and add an equal volume of -20°C precooled isopropanol, invert 3-5 times, and place in a -20°C refrigerator to precipitate for more than 2 hours;

[0071] (5) Centrifuge at 4°C for 15 min, wash the DNA precipitate twice with 70% ethanol, air-dry, dissolve in TE, and store at 4°C until use;

[0072] (6) DNA quality was measured using a UV spectrophotometer, with an OD260 / OD280 ratio ranging from 1.8 to 2.0. Agarose gel electrophoresis was used for verification, and the bands were clear and had no diffusion.

[0073] 3. SSR primer development

[0074] (1) Based on the obtained grape genome data, the MicroSAtellite Identification tool (MISA) was used to develop SSR marker loci. The search parameters were: dinucleotide repeats 6 or more; trinucleotide repeats 5 or more; tetranucleotide repeats 5 or more; pentanucleotide repeats 5 or more; hexanucleotide repeats 5 or more.

[0075] (2) Primer5.0 software was used to design SSR primers. The default parameters were: primer length range 18-25 bp, annealing temperature 58-62°C, GC content 40%-60%, product length 100-250 bp. Primer synthesis was completed by General Biotech Co., Ltd.

[0076] 4. PCR amplification

[0077] Eight samples of grape genomic DNA were randomly selected as amplification templates, and the designed primers were preliminarily screened. Gradient PCR was used to explore the optimal annealing temperature and amplification efficiency of the primers, and then they were used for sample amplification.

[0078] (1) The PCR amplification system was as follows: ddH2O 2 μL, 2× Taq master mix 5 μL, 20 ng / μL template DNA 1 μL, 10 μM forward primer and 10 μM reverse primer 1 μL each.

[0079] (2) The PCR amplification procedure included: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; and extension at 72°C for 10 min.

[0080] 5. SSR marker screening

[0081] (1) 8% polyacrylamide gel electrophoresis (PAGE)

[0082] a. Gel system: ddH2O 30mL, 30% gel-making solution 15mL, 10x TBE 5mL, 10% AP 380μL, TEMED 40μL.

[0083] b. Electrophoresis operation program: voltage 200V, current 400mA, 1.3h.

[0084] c. Development (silver staining): After electrophoresis, staining is performed, the gel plates are separated, the bottom plate is rinsed with distilled water, the gel is taken out, and placed in 0.1% AgNO3 staining solution, and shaken in parallel for 10 minutes; after silver staining, color development is performed, the bottom plate is taken out and rinsed with distilled water twice, after rinsing, the gel is placed in 1.5% NaOH color developing solution containing formaldehyde, and shaken in parallel until color is developed, about 4 minutes; after color development, the gel is rinsed in clean water twice, and the band can be read after rinsing.

[0085] (2) Based on the electrophoresis results, primers with clear bands at the same mobility position on the gel were selected for statistical analysis. Primers with bands were recorded as "1" and primers without bands were recorded as "0".

[0086] (3) Each band of each primer was regarded as a specific site. Primers with clear bands and polymorphism among varieties were selected and sorted from low to high according to primer polymorphism to develop a specific SSR marker combination for the variety resource sample set.

[0087] Table 2: Primers of 10 selected SSR markers and ranking of 14 specific loci

[0088]

[0089] 6. Construction of molecular ID cards for grape varieties

[0090] (1) Perform PCR amplification and detection on the genomic DNA of all samples according to the SSR molecular marker combination obtained by screening;

[0091] (2) 8% polyacrylamide gel electrophoresis was used to detect the PCR product. The gel electrophoresis results were as follows: Figure 2 ;

[0092] (3) Using Excel to collect statistics on the DNA fingerprint information of each sample in the variety resource library and generate a 0-1 matrix;

[0093] (4) Use the Excel data merging formula (concat) to convert the 0-1 matrix into the DNA molecule ID number of each sample.

[0094] Table 3: 0-1 matrix after DNA fingerprint conversion of 50 materials tested

[0095]

[0096]

[0097] Table 4: Molecular ID data table of 50 grape varieties

[0098]

[0099]

[0100] Example 2 Application of grape variety molecular ID data table to unknown sample identification

[0101] It is used to identify unknown varieties in the germplasm resource bank and test 5 unknown samples:

[0102] 1. Use the CTAB method to extract the genomic DNA of the sample to be tested (the specific method is the same as above).

[0103] 2. Perform PCR amplification on the DNA of the sample to be tested using primers SEQ ID No: 1-20 with specific site numbers.

[0104] (1) PCR amplification system: ddH2O 2μL, 2×Taq master mix 5μL, 20ng / μL template DNA 1μL, 10μM forward primer and 10μM reverse primer 1μL each.

[0105] (2) PCR amplification program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 10 min.

[0106] 3. Perform 8% polyacrylamide gel electrophoresis (PAGE) test. The electrophoresis results are shown in Figure 3 .

[0107] (1) Gel system: ddH2O 30 mL, 30% gel-making solution 15 mL, 10x TBE 5 mL, 10% AP 380 μL, TEMED 40 μL.

[0108] (2) Electrophoresis operation program: voltage 200 V, current 400 mA, 1.3 h.

[0109] (3) Development (silver staining): After electrophoresis, staining is performed, the gel plates are separated, the bottom plate is rinsed with distilled water, the gel is removed, and placed in 0.1% AgNO3 staining solution, and shaken in parallel for 10 minutes; after silver staining, color development is performed, the bottom plate is removed and rinsed with distilled water twice, and after rinsing, the gel is placed in 1.5% NaOH color developing solution containing formaldehyde and shaken in parallel until color is developed, which takes about 4 minutes; after color development, the gel is rinsed in clean water twice, and the band can be read after rinsing.

[0110] 4. Use Excel to statistically analyze the DNA fingerprint information of the five samples tested in order of specific sites and construct a 0-1 matrix.

[0111] 5. Fill in the 0-1 matrix information of the five samples to be tested in sequence below the constructed 0-1 matrix of the variety resource database to generate a merged 0-1 matrix.

[0112] Table 5: DNA fingerprint 0-1 matrix of the samples to be tested and the 50 materials to be tested

[0113]

[0114]

[0115] 6. Use the HeatMap module of TBtools to import the merged 0-1 matrix in the form of a two-dimensional array (vertical axis - sample name, horizontal axis - specific site) to generate a HeatMap heat map to achieve rapid visualization of the matrix (variety DNA identity number information).

[0116] 7. Use HeatMap's hierarchical clustering method to cluster the sample data points, select Euclidean to calculate the genetic distance between samples, and use the longest distance method (Complete) for hierarchical clustering analysis to achieve rapid visual matching of the DNA identity number information data of the sample to be tested and the sample in the variety resource library. The grape germplasm that completely matches the sample in the variety resource library is the identification result of the unknown variety to be tested. X1 is No. 50 Giant Rose, X2 is No. 6 Queen Akun, X3 is No. 42 Red Banana, X4 is No. 39 Black Vannesico, and X5 is No. 21 Crisp Red. The HeatMap identification results are shown in Figure 5 .

[0117] Example 3 Application of grape variety molecular ID data table to distinguish mixed samples

[0118] It is used to distinguish the mixed samples in the germplasm resource bank and test the 5 mixed samples (Y1, Y2, Y3, Y4, and Y5) (the 5 samples are known to be 3-Aiblin, 18-Bulesha, and 33-Hei Aolin):

[0119] 1. Use the CTAB method to extract the genomic DNA of the sample to be tested (the specific method is the same as above).

[0120] 2. Perform PCR amplification on the confounded sample DNA using primers SEQ ID No: 1~20 with specific site numbers.

[0121] (1) PCR amplification system: ddH2O 2μL, 2×Taq master mix 5μL, 20ng / μL template DNA 1μL, 10μM forward primer and 10μM reverse primer 1μL each.

[0122] (2) PCR amplification program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 10 min.

[0123] 3. Perform 8% polyacrylamide gel electrophoresis (PAGE) test. The electrophoresis results are shown in Figure 3 .

[0124] (1) Gel system: ddH2O 30 mL, 30% gel-making solution 15 mL, 10x TBE 5 mL, 10% AP 380 μL, TEMED 40 μL.

[0125] (2) Electrophoresis operation program: voltage 200 V, current 400 mA, 1.3 h.

[0126] (3) Development (silver staining): After electrophoresis, staining is performed, the gel plates are separated, the bottom plate is rinsed with distilled water, the gel is removed, and placed in 0.1% AgNO3 staining solution, and shaken in parallel for 10 minutes; after silver staining, color development is performed, the bottom plate is removed and rinsed with distilled water twice, and after rinsing, the gel is placed in 1.5% NaOH color developing solution containing formaldehyde and shaken in parallel until color is developed, which takes about 4 minutes; after color development, the gel is rinsed in clean water twice, and the band can be read after rinsing.

[0127] 4. Use Excel to count the DNA fingerprint information of the samples tested in order of specific sites, and add the DNA fingerprint information of known varieties to construct a 0-1 matrix, such as Figure 6 shown.

[0128] 5. Use Excel conditional formatting to set a two-color scale staining rule for the 0-1 matrix. Set value (1) and value (0) to different colors. You can directly observe the differences in DNA fingerprints between samples, such as Y1, Y3, and Y4 are the same sample.

[0129] 6. Use the HeatMap module of TBtools to import the 0-1 matrix in the form of a two-dimensional array (vertical axis - sample name, horizontal axis - specific site) to generate a HeatMap heat map, and realize rapid visualization of the matrix (variety DNA identity number information).

[0130] 7. Use HeatMap's hierarchical clustering method to cluster the sample set data points, such as Figure 5 As shown, Y1, Y3, and Y4 are all No. 42 Black Olin, Y2 is No. 3 Aiblin, and Y5 is No. 18 Bressa.

[0131] 8. Use Excel-concat to generate the ID card number of the sample to be tested for auxiliary comparison. Compare it with the ID card numbers of samples 3, 18, and 42. The comparison results are shown in Table 6.

[0132] Table 6: DNA molecular ID numbers of samples to be tested and known samples

[0133]

[0134] 9. If there are a large number of confusing samples and it is not suitable to use Excel for color gradation differentiation, the method in Example 2 can be used for differentiation.

[0135] The embodiments described above only describe the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for identifying unknown grape varieties, characterized in that: Import the 0-1 matrix statistically generated in Excel into TBtools in the form of a two-dimensional array to construct a HeatMap hierarchical clustering heat map for visual variety identification, as follows: (1) Using the MISA tool, SSR primer sequences were randomly designed and developed based on the whole grape genome; (2) Collecting young leaves of grape variety resources as plant materials and extracting sample genomic DNA; (3) Randomly select 8-10 sample DNAs as amplification templates, use the SSR primers in step (1) for PCR amplification, and preliminarily screen the primers to select primers with clear bands and polymorphism among varieties. Each band of each pair of primers is used as a specific site. The selected primer sequences are shown in SEQ ID No: 1-20; (4) Using the primer combinations shown in SEQ ID No: 1-20, PCR detection was performed on the grape variety resource samples in sequence; (5) Detect the PCR amplification results by polyacrylamide gel electrophoresis to obtain the DNA fingerprint of grape variety resources; (6) Convert the polymorphic bands in the DNA fingerprint of the variety resources into a 0-1 matrix: the rows and columns are different samples, and the vertical columns are the sorted primer sites. The target band information amplified from different varieties at different sites is counted, where the presence of a band is recorded as "1" and the absence of a band is recorded as "0"; (7) Use the Excel-concat data merging formula to connect the constructed 0-1 matrix rows and columns to obtain the resource molecular ID number of the corresponding variety; (8) Use the CTAB method to extract genomic DNA of the sample to be tested; (9) Performing PCR amplification on the genomic DNA of the unknown sample using the primer sequences shown in SEQ ID No: 1-20; (10) The PCR amplification product was detected by 8% polyacrylamide gel electrophoresis, and the electrophoresis results were counted. The 0-1 matrix information of the unknown samples to be detected was added to the data table in sequence according to the 0-1 matrix construction method; (11) Use TBtools software, select the HeatMap heat map tool in the HeatMap Illustrator module in Graphics, input the two-dimensional array format information of the 0-1 matrix constructed in step (3), click start, get preliminary results, select Control Dialog for heat map analysis, select Euclidean distance for Dist Method, calculate the genetic distance between samples, select the longest distance method for Cluster Method to perform hierarchical clustering analysis, and the cluster heat map is presented, realizing hierarchical clustering with the molecular ID number of each sample as the data point; (12) Analyze the results of the HeatMap clustering heat map: If the unknown sample to be tested is clustered with one of the samples in the variety resource library and the DNA fingerprint information is completely matched, the real variety name of the unknown sample is the corresponding variety of the sample; The grape variety is selected from one or more of Aligot, Armas, Aibel, Elwein, Iona, Ayun Queen, Pinot Blanc, White Collet, White Banana, White Sheshi, White Feather, Baichel, Beisaihun, Bud Rose, Green Pearl, Pollux, Bugisuri, Bresa, Cabernet Sauvignon, Crisp Peak, Crisp Red, Dabao, Dilawa, Emerald Rose, Fengbao, Fenghou, Fengshou, Fusling, Takasago, Guifei Rose, Guirenxiang, Hamilak, Black Olin, Kuroshio, Black Peak, Black Rose, Black Queen, Black Sebil, Black Vanisico, Red Igawa, Red Shuangwei, Red Banana, Red Yi, Pinot Gris, Venus Seedless, Igawa, Wine White, Kyoho, Giant Whale and Giant Rose.

2. A method for distinguishing confusing grape varieties, characterized in that: Excel directly compares the DNA fingerprint information of known varieties and confused varieties and combines the HeatMap hierarchical clustering heat map to perform visual variety identification, as follows: (1) Using the MISA tool, SSR primer sequences were randomly designed and developed based on the whole grape genome; (2) Collecting young leaves of grape variety resources as plant materials and extracting sample genomic DNA; (3) Randomly select 8-10 sample DNAs as amplification templates, use the SSR primers in step (1) for PCR amplification, and preliminarily screen the primers to select primers with clear bands and polymorphism among varieties. Each band of each pair of primers is used as a specific site. The selected primer sequences are shown in SEQ ID No: 1-20; (4) Using the primer combinations shown in SEQ ID No: 1-20, PCR detection was performed on the grape variety resource samples in sequence; (5) Detect the PCR amplification results by polyacrylamide gel electrophoresis to obtain the DNA fingerprint of grape variety resources; (6) Convert the polymorphic bands in the DNA fingerprint of the variety resources into a 0-1 matrix: the rows and columns are different samples, and the vertical columns are the sorted primer sites. The target band information amplified from different varieties at different sites is counted, where the presence of a band is recorded as "1" and the absence of a band is recorded as "0"; (7) Use the Excel-concat data merging formula to connect the constructed 0-1 matrix rows and columns to obtain the resource molecular ID number of the corresponding variety; (8) Use the CTAB method to extract genomic DNA of the sample to be tested; (9) Perform PCR amplification on the genomic DNA of the confounding sample using the primer sequences shown in SEQ ID No: 1-20; (10) The PCR amplification products were detected by 8% polyacrylamide gel electrophoresis, and the electrophoresis results were statistically analyzed to construct a 0-1 matrix of the samples to be tested. The DNA fingerprint information of known varieties was added to this matrix to form a new 0-1 matrix; (11) Use Excel conditional formatting to set a two-color scale staining rule for the 0-1 matrix containing the samples to be tested constructed in step (3), and set the value 1 and the value 0 to different colors to visually distinguish the differences in the sample DNA fingerprints. If two or more colors in the confused varieties are completely consistent, then the confused varieties are the same variety; (12) Use Excel-concat to develop its molecular ID number, perform differential sample comparison, and achieve auxiliary differentiation: if the ID number of the variety to be tested is exactly the same as that of the known variety, then the sample to be tested is the corresponding known variety; The grape variety is selected from one or more of Aligot, Armas, Aibel, Elwein, Iona, Ayun Queen, Pinot Blanc, White Collet, White Banana, White Sheshi, White Feather, Baichel, Beisaihun, Bud Rose, Green Pearl, Pollux, Bugisuri, Bresa, Cabernet Sauvignon, Crisp Peak, Crisp Red, Dabao, Dilawa, Emerald Rose, Fengbao, Fenghou, Fengshou, Fusling, Takasago, Guifei Rose, Guirenxiang, Hamilak, Black Olin, Kuroshio, Black Peak, Black Rose, Black Queen, Black Sebil, Black Vanisico, Red Igawa, Red Shuangwei, Red Banana, Red Yi, Pinot Gris, Venus Seedless, Igawa, Wine White, Kyoho, Giant Whale and Giant Rose.

3. The method according to claim 1 or 2, characterized in that: In the step (2), the genomic DNA extraction method is the CTAB method: the young grape leaves are placed in liquid nitrogen and ground completely, and then the genomic DNA is extracted by the CTAB method and diluted to a concentration of 20 ng / μL.

4. The method according to claim 1 or 2, characterized in that In step (3), the PCR amplification system, based on 10 μL, includes: 2 μL ddH2O, 5 μL 2×Taq master mix, 1 μL 20 ng / μL template DNA, 1 μL each of 10 μM forward primer and 10 μM reverse primer; the PCR amplification program is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 10 min.

5. The method according to claim 1 or 2, characterized in that In step (5), 8% polyacrylamide gel is used for gel electrophoresis, and the 8% polyacrylamide gel preparation includes: 30 mL of ddH2O, 15 mL of 30% gel preparation solution, 5 mL of 10×TBE, 80 μL of 10% AP3, and 40 μL of TEMED; the gel electrophoresis program is: voltage 200 V, current 400 mA, and time 1.3 h; the gel color is developed using the silver staining method.

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