Method for establishing fingerprints and molecular identity cards of citrus grandis based on SSR (Simple Sequence Repeat)

Abstract

The invention discloses a method for establishing a fingerprint spectrum and a molecular identity card of citrus grandis based on SSR (Simple Sequence Repeat), and belongs to the technical field of molecular biology. According to the invention, 10 pairs of SSR core primers with good polymorphism and clear bands are screened out by using agarose gel electrophoresis, denaturing polyacrylamide gel electrophoresis and fluorescent capillary electrophoresis, the variety of the citrus reticulata blanco is detected, 51 citrus reticulata blanco can be completely distinguished, and fingerprint spectrums and molecular identification cards of 51 citrus reticulata blanco are constructed. The method can be used for analyzing genetic evolution, classification status and genetic relationship of the broad-peel citrus, and can efficiently identify wild varieties and cultivated varieties of the broad-peel citrus. The method can effectively avoid homonymous foreign matter and same-object different-name conditions of the citrus reticulata blanco, can be applied to construction of a citrus reticulata blanco germplasm resource garden and production and popularization of nursery stocks, ensures uniqueness of varieties, and assists in healthy and high-quality development of citrus reticulata blanco industry.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for establishing a fingerprint spectrum and a molecular identity card of a broad-skinned citrus fruit based on SSR. Background Art

[0002] Citrus is one of the world's most important fruits, cultivated in over 100 countries, including tropical and subtropical regions between 18°5' and 33°43' north latitude. Citrus tangerines, particularly broad-skinned mandarins, are second only to sweet oranges in terms of fresh consumption in my country, possessing both economic and scientific value. China is the world's oldest country cultivating citrus, with a history dating back to the Xia and Yu eras before Christ. Since the 21st century, China's citrus planting area has increased significantly, ranking first globally.

[0003] China is one of the major origins and diversity centers of citrus plants, boasting a rich variety resource. Citrus is a perennial fruit tree with extensive interspecific and intergeneric hybridization, resulting in a rich citrus resource and a complex genetic background. Citrus germplasm resources include cultivated varieties, wild species, and variants. The extensive cross-compatibility between species, high bud mutation rates, and scarcity of wild resources present challenges in citrus taxonomy, identification, and breeding.

[0004] Corresponding research has been conducted, such as the Chinese patent application No. 201010513720.X citrus variety standard DNA fingerprint library and its construction method, and the citrus variety standard DNA fingerprint library, which is obtained according to the steps of extracting total DNA of each citrus variety, screening SSR characteristic primer pair sequences, obtaining citrus DNA characteristic fingerprint maps and constructing the citrus standard DNA fingerprint library; the application of the invented citrus standard DNA fingerprint library to identify citrus varieties has the advantages of good repeatability, stability and reliability, and rapid detection, which provides a basis for using SSR molecular marker technology to identify citrus varieties. The invented citrus standard DNA fingerprint library can be directly applied to the authenticity identification of citrus varieties, solving the long-standing problem of early identification of citrus seedlings, so that citrus variety identification is no longer restricted by time and space conditions.

[0005] Strengthening the collection, preservation, and identification of citrus germplasm resources is of great significance. Citrus variety identification is difficult using morphology for varieties with very similar leaf morphologies. Using modern molecular biology techniques to identify citrus varieties at the DNA molecular level is more convenient, accurate, and reliable. PAGE, on the other hand, is cumbersome, uses toxic reagents, has low detection efficiency, and cannot accurately identify fragment sizes.

[0006] SSR markers are characterized by rich polymorphism, good reproducibility, and co-dominance. Fluorescence capillary electrophoresis (FCE) overcomes the shortcomings of traditional identification methods and provides stable and reliable amplified fragment sizes. Currently, SSR FCE is rarely used in citrus. Therefore, constructing a DNA molecular fingerprint and molecular ID for citrus based on SSR FCE is of great significance for the standardization of citrus DNA molecular fingerprints. Summary of the Invention

[0007] In view of the problem of lack of standardization of citrus DNA molecular fingerprints in the prior art, the present invention provides a method for establishing a fingerprint map and molecular ID card of wide-peeled citrus based on SSR. The present invention uses agarose gel electrophoresis, denaturing polyacrylamide gel electrophoresis, and fluorescence capillary electrophoresis to screen out 10 pairs of SSR core primers with good polymorphism and clear bands to detect wide-peeled citrus varieties. 51 samples of wide-peeled citrus can be completely distinguished, and fingerprint maps and molecular ID cards of 51 samples of wide-peeled citrus are constructed. It can be used to analyze the genetic evolution, taxonomic status, and kinship of wide-peeled citrus, and can efficiently identify wild varieties and cultivated varieties of wide-peeled citrus. It can effectively avoid the situation of wide-peeled citrus with the same name or the same thing with different names, and can be applied to the construction of wide-peeled citrus germplasm resource gardens and the production and promotion of seedlings to ensure the uniqueness of the varieties and contribute to the healthy and high-quality development of the citrus industry.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A method for establishing a fingerprint and molecular ID card of citrus tangerine based on SSR, comprising the following steps:

[0010] S1. DNA extraction:

[0011] Collect fresh citrus leaves, take 5 leaves, wash and dry them, remove the veins, weigh 0.1 g and place them in a 2 mL centrifuge tube, and store the remaining leaves in a -80°C refrigerator until use. DNA was extracted using the modified CTAB method;

[0012] S2. Determination of DNA quality and concentration:

[0013] The quality of the extracted DNA was tested by 1.2% agarose gel electrophoresis. 5 μL of the DNA stock solution was added to 1 μL of 6× Loading Buffer and mixed evenly. 4 μL of the marker molecular weight standard was applied at 120V for 30 minutes. If there were no bright streaks or dragging in the spotted wells, it indicated that the extracted DNA was not degraded and contained no impurities such as proteins, indicating good extraction quality. The A260 / A280 ratio and DNA content were determined using a nucleic acid protein quantitative analyzer. 1 μL of the DNA stock solution was measured using a nucleic acid protein quantitative analyzer. An A260 / A280 ratio of 1.8-2.0 indicated appropriate purity for the experiment.

[0014] S3. Search for SSR loci:

[0015] The genome sequence of Clementine mandarin was downloaded from NCBI. The SSR loci in the whole genome of Clementine mandarin were searched and counted using MISA. The search criteria for SSR loci were as follows: the number of repeats of mononucleotide (Mono-), dinucleotide (Di-), trinucleotide (Tri-), tetranucleotide (Tetra-), pentanucleotide (Penta-), and hexanucleotide (Hexa-) was set to be greater than or equal to 6, 5, 5, 5, and 5, respectively, and single base repeat units and compound repeat units were removed.

[0016] S4. Primer design and synthesis:

[0017] Through the analysis of genomic SSR loci, appropriate loci were selected and primers were designed based on the flanking sequences (±500 bp) on both sides of the SSR loci. SSR primers were designed using Premier 5.0 software. The primer design requirements are as follows:

[0018] S4-1, primer length: 20-24 bp;

[0019] S4-2, GC content of primer sequence: 40%-60%;

[0020] S4-3, annealing temperature: 57-62°C;

[0021] S4-4, product size: 100-350 bp;

[0022] S5. PCR amplification of citrus:

[0023] S5-1. Determination of primer annealing temperature: Using tangerines as DNA amplification templates, perform gradient PCR on the designed primer annealing temperatures. Using the average sum of the forward and reverse primer Tm values ​​as the critical value, perform PCR amplification at three temperatures within ±5°C. Detection by agarose gel electrophoresis determines the optimal annealing temperature for each primer pair.

[0024] S5-2. Conventional PCR amplification system and procedures:

[0025] S5-2-1. Reaction system: The total reaction system is 25 μL, and the components are as follows: 2 μL dNTP, 1 μL F (forward primer), 1 μL R (reverse primer), 0.2 μL thermostable DNA polymerase, 2.5 μL 10× Buffer, and 1 μL DNA template.

[0026] S5-2-2. Amplification procedure: The reaction procedure was as follows: 94°C pre-denaturation for 5 min, 94°C denaturation for 45 s, 56-62°C annealing for 45 s, 35 cycles; 72°C extension for 1 min, 72°C for 10 min, and storage at 4°C.

[0027] S5-3, SSR fluorescence PCR amplification:

[0028] S5-3-1. Reaction system: 10 μL PCR reaction system, including: 5.0 μL 2× TaqPCR MasterMix, forward and reverse primers (10 mol·L -1 ) 0.5 L each, genomic DNA (20 ng·μL -1 ), ultrapure water 3.0 μL;

[0029] S5-3-2. Reaction procedure: PCR reaction procedure: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 62°C-52°C annealing for 30 s, 35 cycles; 72°C extension for 30 s, 72°C extension for 20 min, storage at 4°C;

[0030] S6. 2% agarose gel electrophoresis detection - primer screening:

[0031] Six distinct citrus varieties were used for primer screening: Shatang orange, Newhall navel orange, Shatian pomelo, Eureka lemon, Golden bullet, and Citrus trifoliata. Each reaction was replicated twice, and the 2% agarose gel electrophoresis assay was performed as follows:

[0032] S6-1. Clean the rubber sheet and comb, dry them and assemble them;

[0033] S6-2. Weigh 1.8 g of agarose and pour it into a wide-mouth bottle containing 150 ml of TBE buffer;

[0034] S6-3. Heat in a microwave oven until completely dissolved.

[0035] S6-4. After complete dissolution, add 7.5 μL of nucleic acid dye and stir evenly on a magnetic stirrer;

[0036] S6-5, wait for the temperature to drop, pour into the plastic sheet at a constant speed, and let it stand at room temperature for 30 minutes;

[0037] S6-6, add 5 μL of 6× Loading Buffer to the PCR amplification product, vortex to mix, add 6 μL to the sample well, and spot 4 μL of the molecular weight marker;

[0038] S6-7, 130V, electrophoresis for 60min;

[0039] S6-8. After the electrophoresis is completed, place the gel into the gel imaging system and take pictures for preservation;

[0040] S7. 6% denaturing polyacrylamide gel electrophoresis detection - primer rescreening:

[0041] Primers with clear, single, and uncontaminated bands obtained by initial screening with 2% agarose gel electrophoresis were selected for further screening with 6% denaturing polyacrylamide gel electrophoresis. Each reaction was repeated twice. The steps for 6% denaturing polyacrylamide gel electrophoresis detection were as follows:

[0042] S7-1. Cleaning the glass plates: First, clean the long and short glass plates with detergent, then rinse with ultrapure water, and then clean the long and short glass plates for glue making with 95% alcohol three times;

[0043] S7-2. Processing short and long glass plates: Spray stripping silane evenly on the short adhesive glass plate, wipe it with dust-free lens cleaning paper, apply it three times and wipe it dry; put on a new pair of gloves, evenly apply adhesive adhesion liquid on the inside of the long glass plate, wipe it with lens cleaning paper, apply it three times and wipe it dry;

[0044] S7-3. Assemble the glass plates: Use the horizontal glue making method to place the long glass plate on the right side of the glue pouring rack and the short glass plate on the left side of the horizontal glue pouring rack to form a prefabricated glue cavity (push the glass plates from left to right). Use a pipette to draw sterile water and evenly apply it on the edge of the long glass plate. Attach side gaskets (to prevent the side gaskets of the glass plates from sliding during glue pouring).

[0045] S7-4. Prepare the solution according to the formula. Add tetramethylethylenediamine (TEMED) and ammonium persulfate (AP) in a fume hood (the drugs are toxic). Shake well. The formula for 6% denatured polyacrylamide gel is as follows:

[0046]

[0047] S7-5, glue pouring: Pour the reagents in the table into the beaker according to the amount, finally add tetramethylethylenediamine (TEMED) and ammonium persulfate (AP), mix well, and use a syringe to draw up the mixture. With your left hand, push the short glass plate forward on the long glass plate to form a glue pouring cavity. With your right hand, inject the gel liquid into the glue pouring cavity (inject at a constant speed to avoid bubbles).

[0048] S7-6, insert the comb: After the glue is poured, insert the comb in time, use the left and right hands to fix the two sides of the glue plate with clamps at the same time, and let it stand for more than 1 hour;

[0049] S7-7. Pre-electrophoresis: Remove the clamps and fix the gel plate formed by the two glass plates to the electrophoresis tank. Add an appropriate amount of 1× TBE buffer to the electrophoresis tank (DNA sequencing electrophoresis tank). Carefully remove the comb and use a syringe without a needle to draw TBE buffer into the gel wells from left to right several times to flush out the urea in the gel wells to prevent overflow of PCR products during spotting and resulting spotting failure. Run the gel at a fixed power of 100 W for 45 minutes to preheat the gel.

[0050] S7-8. Product denaturation: While the gel is preheating, denature the PCR product and marker (DNA molecular weight standard): add an equal volume of 5 μL denaturing dye to 5 μL of each PCR product and marker (DNA molecular weight standard), mix well, incubate at 70°C for 5 min, and immediately place on ice for at least 10 min.

[0051] S7-9. Sample Spotting and Electrophoresis: After preheating, use a large syringe (without a needle) to draw up 1× TBE buffer and flush the wells of the denatured PAGE gel from left to right to rinse out the urea in the wells (to prevent sample spotting failure). Then, add 2 μL of denatured PCR product and 1.5 μL of DNA molecular weight standard to the wells of the gel. Perform electrophoresis at a fixed power of 100 W for 2 h.

[0052] S7-10, Separate the long and short glass plates: After electrophoresis, take out the glass plates, remove the short glass plate (the glue is thin, so be careful not to damage it), and place the long glass plate in the staining frame;

[0053] S7-11, Fixation: After the gel has cooled, place the gel in the fixative solution, place it on a shaker, and shake slowly for 20 minutes. Recover the fixative solution for later use;

[0054] S7-12, rinsing: rinse the gel with ultrapure water 3 times, 2 min each time;

[0055] S7-13, Silver staining: Place the gel in the staining solution and immediately place on a shaker for 30 minutes;

[0056] S7-14, color development: quickly remove the gel from the staining solution and quickly immerse it in ultrapure water. After 1 second, remove the gel and place it in 1000 mL of developer solution. Gently shake it until the developer solution begins to turn gray. Remove the gel and place it in another box containing 1000 mL of developer solution. When gray-brown stripes appear on the gel, immediately pour in 1000 mL of fixative solution and gently shake the mixture for 8 minutes.

[0057] S7-15, rinsing: When the band appears, immediately remove the cleaning gel twice, each time for 2 minutes;

[0058] S7-16, Scan and save: After the gel is dried, place it on a scanner to scan and save the image; select primers that have successfully amplified, have clear polymorphic bands, and contain at least one band for fluorescence capillary electrophoresis detection;

[0059] S8, SSR fluorescence capillary electrophoresis detection:

[0060] Select multiple materials for primer screening;

[0061] S8-1. Synthesis of SSR fluorescent primers: The primers for this experiment use the primers with good polymorphism obtained by polyacrylamide gel electrophoresis screening in the previous step and are labeled with fluorescence for capillary electrophoresis detection. During PCR amplification, the 5' end of the forward primer is labeled with a fluorescent group of different colors, and the reverse primer is synthesized conventionally. When loading the capillary, an adapter sequence is added to the 5' end of the forward primer, and the reverse primer is synthesized conventionally.

[0062] S8-2, Capillary electrophoresis detection - primer screening: dilute the fluorescent PCR product to a uniform concentration before use (to ensure the specificity of fluorescent PCR amplification and the concentration uniformity of the sample on the machine);

[0063] Formamide was mixed with the molecular weight internal standard GeneScan TM Mix thoroughly with 500 LIZ, pipette 9 μL of the mixture into a PCR well plate, add 1 μL of the diluted PCR product, vortex mix thoroughly, denature at 95°C for 5 min, and load the sample onto a 96-channel fully automatic ABI3730XL genetic analyzer for capillary electrophoresis genotyping. Use GeneMarker analysis software to analyze the raw data, compare the position of the molecular weight internal standard in each lane with the position of the peak of each sample, and determine the fragment size and genotype analysis.

[0064] Remove primers that failed to detect at a certain site in some varieties and had no target bands; select sites that amplified bands with good peak patterns and more than 3 alleles for amplification of wide-skinned citrus materials;

[0065] S9. Data analysis:

[0066] After fluorescence capillary electrophoresis, the data were read. The amplified genotypes of homozygous sites were recorded as X / X, and the genotypes of heterozygous sites were recorded as X / Y, where X and Y were the two allelic variants at the site, and the data with smaller amplified molecular weight were presented first, while the data with larger amplified molecular weight were presented last. The polymorphism information content (PIC) and gene diversity (D) of the primers were calculated using Cervus software. On this basis, the observed number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and Shannon diversity index (I) of the primers were calculated using POPGENE version 1.31 software. The genetic distance (Nei's genetic distance D) between each sample was calculated using GenAIex software, and a phylogenetic tree was constructed based on the genetic distance.

[0067] S10. Construction of citrus fingerprint:

[0068] DNA fingerprinting can be constructed by labeling each citrus variety and the alleles amplified by the primers using an Excel spreadsheet, forming an intuitive map. The genotypes of the primers in the sample are directly used to construct the fingerprint map, and the amplified alleles are arranged in ascending molecular weight order and numbered with Arabic numerals (1, 2, 3, 4, 5, 6, 7, 8, 9, etc.). The map is drawn in the form of a spreadsheet, with the horizontal axis representing the molecular weight amplified by each pair of primers and the vertical axis representing different varieties, forming an intuitive DNA fingerprint map of the entire sample.

[0069] S11. Construction of Citrus Molecular Identity Card:

[0070] Amplify the test material to obtain the amplified allele data of each sample, and convert the allele assignment code into a number, namely the molecular identity card (molecular ID);

[0071] The coding method is as follows: the sizes of the amplified fragments (alleles) generated by each pair of primers in the wide-peeled citrus are sorted in ascending order, and then the Arabic numerals 1, 2, 3, ..., 9 are used to encode different amplified band types (genotypes). When the number of band types is greater than 9, capital letters A, B, C are used to represent the 10th, 11th, and 12th amplified band types, and so on. No amplified band is represented by 0; then, in order from the number of band types to the largest number, the codes of the primer markers of the tested germplasm resources are concatenated, thus forming a molecular identity card composed of numbers or letters.

[0072] In the present invention:

[0073] The removal of single-base repeat units and complex repeat units in step S3 is because the sites of single-base repeat units are difficult to detect and interpret accurately. Taq enzyme will insert A at the end of the PCR product, and complex repeat units may also produce different alleles with single-base differences.

[0074] The step S8 of selecting multiple materials for primer fine screening is to select 12 materials for primer fine screening, including: Guposhan Yuanju, Mangshan Wild Ju, Biangan, Shatangju, Ponkan 79-2, Miyamoto, Dazhong Orange, Newhall Navel Orange, Shatian Pomelo, Guang Foshou, Jindan, and Citrus aurantium.

[0075] The fingerprint pattern described in step S10 refers to an electrophoresis pattern that can identify differences between samples.

[0076] Furthermore, step S11 forms a molecular ID card composed of numbers or letters, and converts the molecular ID card information, biological traits and other information into a scannable QR code. By unifying and standardizing each variety to form a unique molecular ID card and converting it into a QR code, variety-related information will appear after scanning, which can accurately represent the identity information of a variety and can be promoted and used in citrus germplasm resource gardens and seedling production.

[0077] Compared with the prior art, the present invention has the following advantages:

[0078] 1. The method of establishing a fingerprint and molecular ID card of citrus serrata based on SSR described in the present invention has 10 pairs of core primers screened with high repeatability, stable results, obvious differences in target bands, easy to judge, and highly compatible with fluorescence capillary electrophoresis.

[0079] 2. The method of establishing the fingerprint and molecular identification of the wide-skinned citrus based on SSR described in the present invention can accurately and efficiently generate the fingerprint and molecular identification of the wide-skinned citrus, which is of great significance for analyzing the genetic evolution, taxonomic status, kinship and identification of the wide-skinned citrus.

[0080] 3. The method of establishing the fingerprint map and molecular ID card of wide-skinned citrus based on SSR described in the present invention can effectively avoid the situation where wide-skinned citrus has the same name but different species or the same species has different names. It can be applied to the construction of wide-skinned citrus germplasm resource gardens and the production and promotion of seedlings to ensure the uniqueness of the varieties and contribute to the healthy and high-quality development of the citrus industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 This is an agarose gel electrophoresis diagram of the initial screening of different primers in the embodiment of the present invention;

[0082] Figure 2 This is a denaturing polyacrylamide gel electrophoresis diagram of multiple screening of different primers in the embodiment of the present invention;

[0083] Figure 3 This is a capillary electrophoresis peak diagram of two pairs of primers amplified by the primer fine screening of the embodiment of the present invention;

[0084] Figure 4 This is the DNA molecular fingerprint of 51 samples of Citrus aurantii in Example 1 of the present invention. DETAILED DESCRIPTION

[0085] The specific implementation of the present invention is further described below with reference to the examples.

[0086] Example:

[0087] A method for establishing a fingerprint and molecular ID card of citrus tangerine based on SSR, comprising the following steps:

[0088] S1. DNA extraction:

[0089] Collect fresh citrus leaves, take 5 leaves, wash and dry them, remove the veins, weigh 0.1 g and place them in a 2 mL centrifuge tube, and store the remaining leaves in a -80°C refrigerator until use. DNA was extracted using the modified CTAB method;

[0090] S2. Determination of DNA quality and concentration:

[0091] The quality of the extracted DNA was tested by 1.2% agarose gel electrophoresis. 5 μL of the DNA stock solution was added to 1 μL of 6× Loading Buffer and mixed evenly. 4 μL of the marker molecular weight standard was applied at 120V for 30 minutes. If there were no bright streaks or dragging in the spotted wells, it indicated that the extracted DNA was not degraded and contained no impurities such as proteins, indicating good extraction quality. The A260 / A280 ratio and DNA content were determined using a nucleic acid protein quantitative analyzer. 1 μL of the DNA stock solution was measured using a nucleic acid protein quantitative analyzer. An A260 / A280 ratio of 1.8-2.0 indicated appropriate purity for the experiment.

[0092] S3. Search for SSR loci:

[0093] The genome sequence of Clementine orange was downloaded from NCBI. The SSR loci in the whole genome of Clementine orange were searched and counted using MISA. The search criteria for SSR loci were as follows: the number of repeats of mononucleotide (Mono-), dinucleotide (Di-), trinucleotide (Tri-), tetranucleotide (Tetra-), pentanucleotide (Penta-), and hexanucleotide (Hexa-) was set to be greater than or equal to 6, 5, 5, 5, 5, and 5, respectively. Single-base repeat units and compound repeat units were removed because the sites of single-base repeat units are difficult to detect and interpret accurately. The Taq enzyme will insert an A at the end of the PCR product. Compound repeat units may also produce different alleles with single-base differences.

[0094] S4. Primer design and synthesis:

[0095] Through the analysis of genomic SSR loci, appropriate loci were selected and primers were designed based on the flanking sequences (±500 bp) on both sides of the SSR loci. SSR primers were designed using Premier 5.0 software. The primer design requirements are as follows:

[0096] S4-1, primer length: 20-24 bp;

[0097] S4-2, GC content of primer sequence: 40%-60%;

[0098] S4-3, annealing temperature: 57-62°C;

[0099] S4-4, product size: 100-350 bp;

[0100] S5. PCR amplification of citrus:

[0101] S5-1. Determination of primer annealing temperature: Using tangerines as DNA amplification templates, perform gradient PCR on the designed primer annealing temperatures. Using the average sum of the forward and reverse primer Tm values ​​as the critical value, perform PCR amplification at three temperatures within ±5°C. Detection by agarose gel electrophoresis determines the optimal annealing temperature for each primer pair.

[0102] S5-2. Conventional PCR amplification system and procedures:

[0103] S5-2-1. Reaction system: The total reaction system is 25 μL, and the components are as follows: 2 μL dNTP, 1 μL F (forward primer), 1 μL R (reverse primer), 0.2 μL thermostable DNA polymerase, 2.5 μL 10× Buffer, and 1 μL DNA template.

[0104] S5-2-2. Amplification procedure: The reaction procedure was as follows: 94°C pre-denaturation for 5 min, 94°C denaturation for 45 s, 56-62°C annealing for 45 s, 35 cycles; 72°C extension for 1 min, 72°C for 10 min, and storage at 4°C.

[0105] S5-3, SSR fluorescence PCR amplification:

[0106] S5-3-1. Reaction system: 10 μL PCR reaction system, including: 5.0 μL 2× TaqPCR MasterMix, forward and reverse primers (10 mol·L -1 ) 0.5 L each, genomic DNA (20 ng·μL -1 ), ultrapure water 3.0 μL;

[0107] S5-3-2. Reaction procedure: PCR reaction procedure: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 62°C-52°C annealing for 30 s, 35 cycles; 72°C extension for 30 s, 72°C extension for 20 min, storage at 4°C;

[0108] S6. 2% agarose gel electrophoresis detection - primer screening:

[0109] Six different citrus materials were selected for primer screening. Each reaction was repeated twice. The steps for 2% agarose gel electrophoresis were as follows:

[0110] S6-1. Clean the rubber sheet and comb, dry them and assemble them;

[0111] S6-2. Weigh 1.8 g of agarose and pour it into a wide-mouth bottle containing 150 ml of TBE buffer;

[0112] S6-3. Heat in a microwave oven until completely dissolved.

[0113] S6-4. After complete dissolution, add 7.5 μL of nucleic acid dye and stir evenly on a magnetic stirrer;

[0114] S6-5, wait for the temperature to drop, pour into the plastic sheet at a constant speed, and let it stand at room temperature for 30 minutes;

[0115] S6-6, add 5 μL of 6× Loading Buffer to the PCR amplification product, vortex to mix, add 6 μL to the sample well, and spot 4 μL of the molecular weight marker;

[0116] S6-7, 130V, electrophoresis for 60min;

[0117] S6-8. After the electrophoresis is completed, place the gel into the gel imaging system and take pictures for preservation;

[0118] S7. 6% denaturing polyacrylamide gel electrophoresis detection - primer rescreening:

[0119] Primers with clear, single, and uncontaminated bands obtained by initial screening with 2% agarose gel electrophoresis were selected for further screening with 6% denaturing polyacrylamide gel electrophoresis. Each reaction was repeated twice. The steps for 6% denaturing polyacrylamide gel electrophoresis detection were as follows:

[0120] S7-1. Cleaning the glass plates: First, clean the long and short glass plates with detergent, then rinse with ultrapure water, and then clean the long and short glass plates for glue making with 95% alcohol three times;

[0121] S7-2. Processing short and long glass plates: Spray stripping silane evenly on the short adhesive glass plate, wipe it with dust-free lens cleaning paper, apply it three times and wipe it dry; put on a new pair of gloves, evenly apply adhesive adhesion liquid on the inside of the long glass plate, wipe it with lens cleaning paper, apply it three times and wipe it dry;

[0122] S7-3. Assemble the glass plates: Use the horizontal glue making method to place the long glass plate on the right side of the glue pouring rack and the short glass plate on the left side of the horizontal glue pouring rack to form a prefabricated glue cavity (push the glass plates from left to right). Use a pipette to draw sterile water and evenly apply it on the edge of the long glass plate. Attach side gaskets (to prevent the side gaskets of the glass plates from sliding during glue pouring).

[0123] S7-4. Prepare the solution according to the formula. Add tetramethylethylenediamine (TEMED) and ammonium persulfate (AP) in a fume hood (the drugs are toxic). Shake well. The formula for 6% denatured polyacrylamide gel is as follows:

[0124]

[0125] S7-5, glue pouring: Pour the reagents in the table into the beaker according to the amount, finally add tetramethylethylenediamine (TEMED) and ammonium persulfate (AP), mix well, and use a syringe to draw up the mixture. With your left hand, push the short glass plate forward on the long glass plate to form a glue pouring cavity. With your right hand, inject the gel liquid into the glue pouring cavity (inject at a constant speed to avoid bubbles).

[0126] S7-6, insert the comb: After the glue is poured, insert the comb in time, use the left and right hands to fix the two sides of the glue plate with clamps at the same time, and let it stand for more than 1 hour;

[0127] S7-7. Pre-electrophoresis: Remove the clamps and fix the gel plate formed by the two glass plates to the electrophoresis tank. Add an appropriate amount of 1× TBE buffer to the electrophoresis tank (DNA sequencing electrophoresis tank). Carefully remove the comb and use a syringe without a needle to draw TBE buffer into the gel wells from left to right several times to flush out the urea in the gel wells to prevent overflow of PCR products during spotting and resulting spotting failure. Run the gel at a fixed power of 100 W for 45 minutes to preheat the gel.

[0128] S7-8. Product denaturation: While the gel is preheating, denature the PCR product and marker (DNA molecular weight standard): add an equal volume of 5 μL denaturing dye to 5 μL of each PCR product and marker (DNA molecular weight standard), mix well, incubate at 70°C for 5 min, and immediately place on ice for at least 10 min.

[0129] S7-9. Sample Spotting and Electrophoresis: After preheating, use a large syringe (without a needle) to draw up 1× TBE buffer and flush the wells of the denatured PAGE gel from left to right to rinse out the urea in the wells (to prevent sample spotting failure). Then, add 2 μL of denatured PCR product and 1.5 μL of DNA molecular weight standard to the wells of the gel. Perform electrophoresis at a fixed power of 100 W for 2 h.

[0130] S7-10, Separate the long and short glass plates: After electrophoresis, take out the glass plates, remove the short glass plate (the glue is thin, so be careful not to damage it), and place the long glass plate in the staining frame;

[0131] S7-11, Fixation: After the gel has cooled, place the gel in the fixative solution, place it on a shaker, and shake slowly for 20 minutes. Recover the fixative solution for later use;

[0132] S7-12, rinsing: rinse the gel with ultrapure water 3 times, 2 min each time;

[0133] S7-13, Silver staining: Place the gel in the staining solution and immediately place on a shaker for 30 minutes;

[0134] S7-14, color development: quickly remove the gel from the staining solution and quickly immerse it in ultrapure water. After 1 second, remove the gel and place it in 1000 mL of developer solution. Gently shake it until the developer solution begins to turn gray. Remove the gel and place it in another box containing 1000 mL of developer solution. When gray-brown stripes appear on the gel, immediately pour in 1000 mL of fixative solution and gently shake the mixture for 8 minutes.

[0135] S7-15, rinsing: When the band appears, immediately remove the cleaning gel twice, each time for 2 minutes;

[0136] S7-16, Scan and save: After the gel is dried, place it on a scanner to scan and save the image; select primers that have successfully amplified, have clear polymorphic bands, and contain at least one band for fluorescence capillary electrophoresis detection;

[0137] S8, SSR fluorescence capillary electrophoresis detection:

[0138] 12 materials were selected for primer fine screening;

[0139] S8-1. Synthesis of SSR fluorescent primers: The primers for this experiment use the primers with good polymorphism obtained by polyacrylamide gel electrophoresis screening in the previous step and are labeled with fluorescence for capillary electrophoresis detection. During PCR amplification, the 5' end of the forward primer is labeled with a fluorescent group of different colors, and the reverse primer is synthesized conventionally. When loading the capillary, an adapter sequence is added to the 5' end of the forward primer, and the reverse primer is synthesized conventionally.

[0140] S8-2, Capillary electrophoresis detection - primer screening: dilute the fluorescent PCR product to a uniform concentration before use (to ensure the specificity of fluorescent PCR amplification and the concentration uniformity of the sample on the machine);

[0141] Formamide was mixed with the molecular weight internal standard GeneScan TM Mix thoroughly with 500 LIZ, pipette 9 μL of the mixture into a PCR well plate, add 1 μL of the diluted PCR product, vortex mix thoroughly, denature at 95°C for 5 min, and load the sample onto a 96-channel fully automatic ABI3730XL genetic analyzer for capillary electrophoresis genotyping. Use GeneMarker analysis software to analyze the raw data, compare the position of the molecular weight internal standard in each lane with the position of the peak of each sample, and determine the fragment size and genotype analysis.

[0142] Remove primers that failed to detect at a certain site in some varieties and had no target bands; select sites that amplified bands with good peak patterns and more than 3 alleles for amplification of wide-skinned citrus materials;

[0143] S9. Data analysis:

[0144] After fluorescence capillary electrophoresis, the data were read. The amplified genotypes of homozygous sites were recorded as X / X, and the genotypes of heterozygous sites were recorded as X / Y, where X and Y were the two allelic variants at the site, and the data with smaller amplified molecular weight were presented first, while the data with larger amplified molecular weight were presented last. The polymorphism information content (PIC) and gene diversity (D) of the primers were calculated using Cervus software. On this basis, the observed number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and Shannon diversity index (I) of the primers were calculated using POPGENE version 1.31 software. The genetic distance (Nei's genetic distance D) between each sample was calculated using GenAIex software, and a phylogenetic tree was constructed based on the genetic distance.

[0145] S10. Construction of fingerprint of broad-skinned citrus:

[0146] A fingerprint is an electrophoretic pattern that can identify differences between samples. DNA fingerprinting can be constructed by labeling each citrus variety and the alleles amplified by the primers using an Excel spreadsheet, creating a visual map. The genotypes of the primers in the sample are directly used to construct the fingerprint. The amplified alleles are arranged in ascending molecular weight order and numbered using Arabic numerals (1, 2, 3, 4, 5, 6, 7, 8, 9, etc.). The map is then drawn in a spreadsheet format, with the horizontal axis representing the molecular weight amplified by each primer pair and the vertical axis representing the different varieties, forming a visual DNA fingerprint of the entire sample.

[0147] S11. Construction of molecular ID card of Citrus aurantium:

[0148] Amplify the test material to obtain the amplified allele data of each sample, and convert the allele assignment code into a number, namely the molecular identity card (molecular ID);

[0149] The coding method is as follows: the sizes of the amplified fragments (alleles) generated by each primer pair in citrus serrata are sorted in ascending order, and then the different amplified band patterns (genotypes) are coded using the units digits 1, 2, 3, ..., 9. When the number of band patterns is greater than 9, the capital letters A, B, and C represent the 10th, 11th, and 12th amplified band patterns, and so on. No amplified band is represented by 0. Then, the codes of the primer markers of the tested germplasm resources are concatenated in ascending order of the number of band patterns to form a molecular identity card composed of numbers or letters.

[0150] The molecular ID information, biological traits and other information are converted into scannable QR codes. By unifying and standardizing each variety, a unique molecular ID card is formed and converted into a QR code. After scanning, variety-related information will appear, which can accurately represent the identity information of a variety and can be promoted and used in citrus germplasm resource gardens and seedling production.

[0151] Results and Summary:

[0152] 1. Primer screening:

[0153] Figure 1 This is the agarose gel electrophoresis diagram of the initial screening of different primers; Figure 1 As shown, 136 pairs of primers were screened using 2% agarose gel electrophoresis;

[0154] Figure 2 is the electrophoresis diagram of denaturing polyacrylamide gel after repeated screening of different primers; Figure 2 As shown, 90 pairs of primers were screened by 6% denaturing polyacrylamide gel electrophoresis;

[0155] Figure 3 This is the capillary electrophoresis peak diagram of two pairs of primers amplified by primer fine screening; Figure 3 As shown in the figure, 10 pairs of core primers were screened out by capillary electrophoresis, namely S01, S11, S13, S17, S18, S21, S73, S76, S85, and S90 (see Table 1).

[0156] Table 110 Core Primer Information

[0157]

[0158] 2. Evaluation of SSR primer polymorphism:

[0159] A total of 18 pairs of SSR primers with good polymorphism and successful amplification were obtained (see Table 2). The amplified fragment size ranged from 122 to 369 bp. A total of 147 alleles were detected, and the average number of alleles amplified by each pair of primers (Na) was 8.16. The number of alleles amplified at each locus ranged from 4 to 18. The more alleles are amplified, the richer the polymorphism of the primers, and the more differences between samples can be reflected. A total of 239 amplified band patterns (genotypes) were detected in 18 markers. The amplified gene band patterns at each marker locus were different. The number of amplified band patterns of different SSR primers varied from 6 to 30. The primer with the most amplified band patterns was S90, with 30 amplified patterns, and it was also the only pair of primers among the 18 pairs of primers with more than 30 amplified genotypes, indicating that this marker can individually identify up to 30 varieties; the primer with the least was S76, with 6 genotypes. The average value of the Shannon information index of the 18 pairs of primers was 1.410. Among the 18 loci, the highest observed heterozygosity (Ho) of the locus was primer S17, which was 0.833; the lowest observed heterozygosity of the locus was primer S28, which was 0.319; the average value of the observed heterozygosity of the locus was 0.532, indicating high genetic diversity of citrus fruit trees. The highest expected heterozygosity (He) of the locus was primer S17, which was 0.836; the lowest expected heterozygosity of the locus was primer S85, which was 0.469. The largest polymorphism information content (PIC) value of the primer was primer S17, which was 0.814, and the primer with the smallest polymorphism was S76, with a value of 0.411. The average polymorphism information content was 0.621. The larger the PIC value, the better the polymorphism degree of the primer. There were 9 pairs of primers with PIC values higher than the average value of 0.621, accounting for 50%; the number of primers with PIC values less than the average value was equal to that of primers higher than the average value, each accounting for 50%. The average value of PIC was higher than 0.5, indicating high-polymorphism primers, and 0.4 < PIC < 0.5 belonged to medium-polymorphism primers, both indicating rich polymorphism information content of the primers.

[0160] 3. SSR characteristic fingerprint information of 51 loose-skin citrus:

[0161] Figure 4 It is the DNA molecular fingerprint map of 51 loose-skin citrus, as Figure 4As shown, among 51 broad-skinned citrus varieties, 29 possess specific alleles. The number of alleles theoretically reflects the number of identifiable varieties. Therefore, based on specific alleles, broad-skinned citrus varieties containing specific markers can be quickly distinguished from other varieties. Each variety possesses a different number of specific alleles (see Table 3). The 51 broad-skinned citrus varieties included both wild and cultivated varieties. Specific alleles were found in 18 marker pairs, not only between citrus varieties but also within citrus groups. With the exception of markers S01, S11, S13, S28, S73, S76, S84, and S85, all other markers possess specific alleles within cultivated citrus varieties. Furthermore, the number of specific alleles in wild citrus varieties is greater than that in cultivated varieties, allowing for differentiation between wild and cultivated varieties based on specific markers. The reason why there are fewer specific markers in cultivated varieties may be that most of the cultivated varieties are bred through bud mutation selection, seedling selection and hybrid breeding, with high genetic similarity, similar background and the presence of highly homologous sequences.

[0162]

[0163] Table 3 Molecular ID cards of 51 citrus fruits

[0164]

[0165] The above description is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this field, it is possible to make several improvements and changes without departing from the creative concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for establishing a fingerprint and molecular ID of citrus fruit based on SSR, characterized by: The steps include: S1. DNA extraction: Collect fresh citrus leaves, take 5 leaves, wash and dry them, remove the veins, weigh 0.1 g and place them in a 2 mL centrifuge tube, and store the remaining leaves in a -80°C refrigerator until use. DNA was extracted using the modified CTAB method; S2. Determination of DNA quality and concentration: The quality of the extracted DNA was tested by 1.2% agarose gel electrophoresis. 5 μL of the DNA stock solution was added to 1 μL of 6× Loading Buffer and mixed evenly. 4 μL of the marker molecular weight standard was applied at 120V for 30 minutes. If there were no bright streaks or dragging in the spotted wells, it indicated that the extracted DNA was not degraded and had no protein impurities, indicating that the extraction quality was good. Use a nucleic acid protein quantitative analyzer to determine the A260 / A280 ratio and DNA content. Pipette 1 μL of DNA stock solution and measure it using a nucleic acid protein quantitative analyzer. An A260 / A280 ratio of 1.8-2.0 indicates DNA of appropriate purity and is suitable for conducting experiments. S3. Search for SSR loci: The genome sequence of Clementine mandarin was downloaded from NCBI. The SSR loci in the whole genome of Clementine mandarin were searched and counted using MISA. The search criteria for SSR loci were as follows: the number of repeats of mononucleotide Mono-, dinucleotide Di-, trinucleotide Tri-, tetranucleotide Tetra-, pentanucleotide Penta-, and hexanucleotide Hexa- was set to be greater than or equal to 6, 5, 5, 5, and 5, respectively. Single base repeat units and compound repeat units were removed. S4. Primer design and synthesis: Through the analysis of genomic SSR loci, appropriate loci were selected and primers were designed within ±500bp of the flanking sequences on both sides of the SSR loci. SSR primers were designed using Premier 5.0 software. The primer design requirements are as follows: S4-1, primer length: 20-24 bp; S4-2, GC content of primer sequence: 40%-60%; S4-3, annealing temperature: 57-62°C; S4-4, product size: 100-350 bp; S5. PCR amplification of citrus: S5-1. Determination of primer annealing temperature: Using tangerines as DNA amplification templates, perform gradient PCR on the designed primer annealing temperatures. Using the average sum of the forward and reverse primer Tm values ​​as the critical value, perform PCR amplification at three temperatures within ±5°C. Detection by agarose gel electrophoresis determines the optimal annealing temperature for each primer pair. S5-2. Conventional PCR amplification system and procedures: S5-2-1. Reaction system: The total reaction system is 25 μL, and the components are as follows: 2 μL dNTP, 1 μL F forward primer, 1 μL R reverse primer, 0.2 μL thermostable DNA polymerase, 2.5 μL 10× Buffer, 1 μL DNA template; S5-2-2. Amplification procedure: The reaction procedure was as follows: 94°C pre-denaturation for 5 min, 94°C denaturation for 45 s, 56-62°C annealing for 45 s, 35 cycles; 72°C extension for 1 min, 72°C for 10 min, and storage at 4°C. S5-3, SSR fluorescence PCR amplification: S5-3-1. Reaction system: 10 μL PCR reaction system, including: 5.0 μL 2× TaqPCR MasterMix, 10 mol·L forward and reverse primers -1 0.5L each, genomic DNA 20ng·μL -1 , ultrapure water 3.0 μL; S5-3-2. Reaction procedure: PCR reaction procedure: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 62°C-52°C annealing for 30 s, 35 cycles; 72°C extension for 30 s, 72°C extension for 20 min, storage at 4°C; S6. 2% agarose gel electrophoresis detection - primer screening: Six distinct citrus varieties were used for primer screening: Shatang orange, Newhall navel orange, Shatian pomelo, Eureka lemon, Golden bullet, and Citrus trifoliata. Each reaction was replicated twice, and the 2% agarose gel electrophoresis assay was performed as follows: S6-1. Clean the rubber sheet and comb, dry them and assemble them; S6-2. Weigh 1.8 g of agarose and pour it into a wide-mouth bottle containing 150 ml of TBE buffer; S6-3. Heat in a microwave oven until completely dissolved. S6-4. After complete dissolution, add 7.5 μL of nucleic acid dye and stir evenly on a magnetic stirrer; S6-5, wait for the temperature to drop, pour into the plastic sheet at a constant speed, and let it stand at room temperature for 30 minutes; S6-6, add 5 μL of 6× Loading Buffer to the PCR amplification product, vortex to mix, add 6 μL to the sample well, and spot 4 μL of the molecular weight marker; S6-7, 130V, electrophoresis for 60min; S6-8. After the electrophoresis is completed, place the gel into the imaging system and take pictures for preservation; S7. 6% denaturing polyacrylamide gel electrophoresis detection - primer rescreening: Primers with clear, single, and uncontaminated bands obtained by initial screening with 2% agarose gel electrophoresis were selected for further screening with 6% denaturing polyacrylamide gel electrophoresis. Each reaction was repeated twice. The steps for 6% denaturing polyacrylamide gel electrophoresis detection were as follows: S7-1. Cleaning the glass plates: First, clean the long and short glass plates with detergent, then rinse with ultrapure water, and then clean the long and short glass plates for glue making with 95% alcohol three times; S7-2. Processing short and long glass plates: Spray stripping silane evenly on the short adhesive glass plate, wipe it with dust-free lens cleaning paper, apply it three times and wipe it dry; put on a new pair of gloves, evenly apply adhesive adhesion liquid on the inside of the long glass plate, wipe it with lens cleaning paper, apply it three times and wipe it dry; S7-3. Assemble the glass plates: Use the horizontal glue making method to place the long glass plate on the right side of the glue pouring rack and the short glass plate on the left side of the horizontal glue pouring rack to form a prefabricated glue cavity. Push the glass plates from left to right, then use a pipette to draw sterile water and evenly apply it on the edge of the long glass plate. Attach side gaskets to prevent them from sliding during glue pouring. S7-4. Prepare the solution according to the recipe. Add tetramethylethylenediamine and ammonium persulfate in a fume hood and shake well. The formula for 6% denatured polyacrylamide gel is as follows: Reagent Volume S7-5, glue pouring: Pour the reagents in the table into the beaker according to the amount, finally add tetramethylethylenediamine and ammonium persulfate, mix well, and draw up the mixture with a syringe. With your left hand, push the short glass plate forward on the long glass plate to form a glue pouring cavity. With your right hand, inject the gel liquid into the glue pouring cavity. Inject at a constant speed to avoid bubbles. S7-6, insert the comb: After the glue is poured, insert the comb in time, use the left and right hands to fix the two sides of the glue plate with clamps at the same time, and let it stand for more than 1 hour; S7-7. Pre-electrophoresis: Remove the clamps and fix the gel plate formed by the two glass plates to the electrophoresis tank. Add an appropriate amount of 1× TBE buffer to the DNA sequencing electrophoresis tank. Carefully remove the comb and use a syringe without a needle to draw TBE buffer into the gel wells from left to right several times to flush out the urea in the gel wells to prevent overflow of PCR products during spotting and resulting in spotting failure. Run the gel at a fixed power of 100 W for 45 minutes to preheat the gel. S7-8. Product denaturation: While the gel is preheating, denature the PCR product and the marker DNA molecular weight standard: add 5 μL of denaturing dye to 5 μL of each PCR product and marker DNA molecular weight standard, mix well, and incubate at 70°C for 5 minutes, then immediately place on ice for at least 10 minutes. S7-9. Sample Spotting and Electrophoresis: After preheating, use a large syringe without a needle to draw up 1× TBE buffer and flush the wells of the denatured PAGE gel from left to right to rinse out the urea in the wells to prevent sample spotting failure. 2 μL of denatured PCR product and 1.5 μL of DNA molecular weight standard are added to the wells of the gel. Electrophoresis is performed at a fixed power of 100 W for 2 h. S7-10, Separate the long and short glass plates: After the electrophoresis is completed, take out the glass plates, remove the short glass plate, and place the long glass plate in the staining frame. S7-11, Fixation: After the gel has cooled, place the gel in the fixative solution, place it on a shaker, and shake slowly for 20 minutes. Recover the fixative solution for later use; S7-12, rinsing: rinse the gel with ultrapure water 3 times, 2 min each time; S7-13, Silver staining: Place the gel in the staining solution and immediately place on a shaker for 30 minutes; S7-14, color development: quickly remove the gel from the staining solution and quickly immerse it in ultrapure water. After 1 second, remove the gel and place it in 1000 mL of developer solution. Gently shake it until the developer solution begins to turn gray. Remove the gel and place it in another box containing 1000 mL of developer solution. When gray-brown stripes appear on the gel, immediately pour in 1000 mL of fixative solution and gently shake the mixture for 8 minutes. S7-15, rinsing: When the band appears, immediately remove the cleaning gel twice, each time for 2 minutes; S7-16, Scan and save: After the gel is dried, place it on a scanner to scan and save the image; select primers that have successfully amplified, have clear polymorphic bands, and contain at least one band for fluorescence capillary electrophoresis detection; S8, SSR fluorescence capillary electrophoresis detection: Select multiple materials for primer screening; S8-1. Synthesis of SSR fluorescent primers: The primers for this experiment use the primers with good polymorphism obtained by polyacrylamide gel electrophoresis screening in the previous step and are labeled with fluorescence for capillary electrophoresis detection. During PCR amplification, the 5' end of the forward primer is labeled with a fluorescent group of different colors, and the reverse primer is synthesized conventionally. When loading the capillary, an adapter sequence is added to the 5' end of the forward primer, and the reverse primer is synthesized conventionally. S8-2, Capillary electrophoresis detection - primer screening: dilute the fluorescent PCR product to a uniform concentration before use to ensure the specificity of fluorescent PCR amplification and the concentration uniformity of the sample on the machine; Formamide was mixed with the molecular weight internal standard GeneScan TM Mix thoroughly with 500 LIZ, pipette 9 μL of the mixture into a PCR well plate, add 1 μL of the diluted PCR product, vortex mix thoroughly, denature at 95°C for 5 min, and load the sample onto a 96-channel fully automatic ABI3730XL genetic analyzer for capillary electrophoresis genotyping. Use GeneMarker analysis software to analyze the raw data, compare the position of the molecular weight internal standard in each lane with the position of the peak of each sample, and determine the fragment size and genotype analysis. Remove primers that failed to detect at a certain site in some varieties and had no target bands; select sites that amplified bands with good peak patterns and more than 3 alleles for amplification of wide-skinned citrus materials; S9. Data analysis: After fluorescence capillary electrophoresis, the data were read. The amplified genotypes of homozygous sites were recorded as X / X, and the genotypes of heterozygous sites were recorded as X / Y, where X and Y were the two allelic variants at the site, and the data with smaller amplified molecular weight were placed first, and the data with larger amplified molecular weight were placed last. The polymorphism information content and gene diversity of the primers were calculated using Cervus software. On this basis, the number of alleles, effective number of alleles, observed heterozygosity, expected heterozygosity, and Shannon diversity index of the primers were calculated using POPGENE version 1.31 software. The genetic distances between samples were calculated using GenAIex software, and a phylogenetic tree was constructed based on the genetic distances. S10. Construction of citrus fingerprint: DNA fingerprinting can be constructed by using an Excel spreadsheet to label each citrus variety and the alleles amplified by the primers, forming an intuitive map. The genotypes of the primers in the sample are directly used to construct the fingerprint map, and the amplified alleles are arranged in ascending order of molecular weight and numbered with Arabic numerals 1, 2, 3, 4, 5, 6, 7, 8, 9, etc. The map is drawn in the form of a spreadsheet, with the horizontal axis representing the molecular weight amplified by each pair of primers and the vertical axis representing different varieties, forming an intuitive DNA fingerprint map of the entire sample. S11. Construction of Citrus Molecular Identity Card: Amplify the test material to obtain the amplified allele data of each sample, and convert the allele assignment code into a number, i.e., the molecular ID card; The coding method is as follows: the sizes of the amplified fragments generated by each pair of primers in the wide-peeled citrus are sorted in ascending order, and then the Arabic numerals 1, 2, 3, ..., 9 are used to encode different amplified band types. When the number of band types is greater than 9, capital letters A, B, C are used to represent the 10th, 11th, and 12th amplified band types, and so on. No amplified band is represented by 0; then, in order from the number of band types to the largest, the codes of the primer markers of the tested germplasm resources are concatenated, thus forming a molecular identity card composed of numbers or letters.

2. The method for establishing a fingerprint and molecular ID card of citrus tangerines based on SSR according to claim 1, characterized in that: The step S8 of selecting multiple materials for primer fine screening is to select 12 materials for primer fine screening.

3. The method for establishing a fingerprint and molecular ID card of Citrus tangerine based on SSR according to claim 1, characterized in that: Step S11 forms a molecular ID card composed of numbers or letters, converts the molecular ID card information, biological characteristics and other information into a scannable QR code, and forms a unique molecular ID card for each variety through unified standardization and converts it into the form of a QR code. After scanning, variety-related information will appear, accurately indicating the identity information of a variety, and promoting its use in citrus germplasm resource gardens and seedling production.

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