A method for constructing a garlic variety fingerprint and application

Abstract

The application discloses a method for accurately identifying garlic varieties and synchronously evaluating alliin content by using structural variation molecular markers. The application obtains a large number of genetic stable large-scale structural variation sites by de novo assembly of a garlic genome and pan-genome construction; identifies functional structural variations of key alliin synthesis genes AsGGT2 and AsFMO11; further constructs a fingerprint system containing 16 typing markers and two alliin functional markers, and establishes a standardized variety identification process. The method only needs agarose gel electrophoresis to complete detection, is simple and easy to operate, low in cost and stable in result, can realize accurate identification of garlic varieties, can indirectly evaluate the content of the core flavor trait alliin, and provides reliable technical support for garlic germplasm resource protection, new variety right protection and market variety standard management.

Description

Technical Field

[0001] This invention relates to a method for constructing fingerprint profiles of garlic varieties based on molecular markers of genomic structural variations, and to an auxiliary evaluation method for the rapid and accurate identification of garlic varieties and for the evaluation of traits related to alliin content. Background Technology

[0002] Garlic (Allium sativum L.) is an important vegetable crop, with an annual planting area exceeding ten million mu. However, the garlic industry currently faces a severe problem of varietal confusion, primarily stemming from two aspects: First, garlic is an asexually propagated crop, and the garlic bulb (clove) serves both as an agricultural product and a reproductive organ, making it highly susceptible to varietal confusion during production and distribution, leading to widespread phenomena of "synonyms for the same species and homonyms for different species." Second, the plant morphology differences among garlic varieties are relatively small, making accurate identification based solely on external morphological characteristics insufficient, further exacerbating the degree of varietal confusion. This situation not only makes germplasm resource tracing difficult but also hinders the effective protection of the identities of renowned local varieties such as Jinxiang garlic, Cangshan four-six-clove garlic, and Ershui early garlic, severely restricting the accurate identification of garlic germplasm resources, the protection of new variety rights, and the standardized management of the market. Therefore, developing an efficient, rapid, and accurate varietal identification technology has become an urgent need for the industry's development.

[0003] Constructing fingerprint profiles for varieties using molecular markers is an effective method for variety identification and is widely used in various crops. Currently, the main types of molecular markers used for fingerprinting include SSR, KASP, SRAP, and RAPD, each with some limitations. SRAP and RAPD were widely used in the early stages, primarily using random primers for DNA fragment amplification, but they suffer from poor stability and high identification error rates. KASP, a molecular marker developed based on SNP variations, relies on expensive quantitative PCR instruments or specialized genotyping systems for detection, limiting its widespread application. SSRs are simple repetitive sequences in the genome. Due to their variable repetition number, SSR markers are generally multi-allelic and possess rich genetic information, making SSR markers a classic technique for constructing fingerprint profiles. However, the technique itself has the following drawbacks: 1) The differences between different SSR alleles are small, requiring high-resolution polyacrylamide gel electrophoresis for differentiation, making detection time-consuming and labor-intensive; 2) Different laboratories and different batches of genetic analysis may have a few base errors in judging the size of the same SSR, leading to difficulties in data standardization and sharing. In addition, some media outlets reported that Xinjiang garlic uses nine SNPs to construct its identity (…). xj.xinhuanet.com / 20251208 / 53808beee8ae4d248 f56d3c72bb6dab4 / c.htmlThis technology also has several drawbacks: 1) SNP markers are prone to reversion mutations; 2) SNP detection relies on high-throughput sequencing, which requires expensive sequencing equipment and generally needs to be outsourced to sequencing companies; 3) The garlic genome is large, making sequencing a single sample expensive, while bioinformatics SNP mining has a long cycle. These drawbacks make it difficult to apply SNP molecular identification methods.

[0004] The garlic genome is extremely large (approximately 16.9 Gb), with repetitive sequences accounting for as much as 91%, which places extremely high demands on the stability of molecular markers used in variety identification. Structural variations, as a genetically stable type of sequence change in the genome, have a significant stability advantage compared to SNPs, which are prone to reversion mutations, and SSRs, whose length may change due to chain slippage during passage. By designing primers for insertion / deletion structural variation fragments, these variations can be detected using PCR amplification. This method is simple to operate and can be rapidly identified using only agarose gel electrophoresis. Furthermore, alliin content, a key trait determining the flavor quality of garlic, is typically detected using liquid chromatography-mass spectrometry (LC-MS), making it difficult to rapidly assess alliin levels across different varieties. Developing functional markers in alliin synthesis-related genes and integrating them into variety fingerprinting would facilitate an indirect and efficient evaluation of this trait. Summary of the Invention

[0005] Purpose of the invention: One purpose of this invention is to provide a method for constructing a fingerprint spectrum of garlic varieties using structural variation molecular markers. Another purpose of this invention is to provide an application for rapid and accurate identification of garlic varieties and auxiliary evaluation of alliin content-related traits using the garlic variety fingerprint spectrum established by this method, thereby solving the problems of poor stability, complex operation, high cost, and inability to simultaneously evaluate quality traits in existing garlic variety identification technologies.

[0006] Technical solution: The method for constructing a fingerprint spectrum of a garlic variety according to the present invention includes the following steps:

[0007] (1) Sample DNA extraction: Leaf tissue samples of several known garlic varieties were collected, and genomic DNA was extracted using the CTAB method;

[0008] (2) PCR amplification: Genomic DNA was amplified by PCR using a specific primer combination. The specific primer combination corresponded to 16 genotyping markers, 2 allicin content functional markers and 1 internal reference marker.

[0009] (3) DNA quality assessment: DNA sample quality is assessed by the internal control label band. If the internal control label has no clear band, the DNA sample is considered to have degraded or failed to be extracted; if the internal control label band is clear, the DNA sample is considered to be qualified.

[0010] (4) Genotype reading and fingerprint pattern construction: For qualified DNA samples, the bands of 16 genotyping markers and 2 functional markers are read in sequence. The amplification product with the corresponding band is counted as "1" and the absence of the corresponding band is counted as "0", thus forming the molecular fingerprint patterns of multiple garlic varieties.

[0011] In step (2), the PCR amplification reaction system consisted of 10 μL standard: 5.0 μL of 2×Taq PCR Master Mix, 0.5 μL of each labeled upstream primer (10 μmol / L), 0.5 μL of each labeled downstream primer (10 μmol / L), 1.0 μL of template DNA (20~50 ng / μL), and 3.0 μL of sterile ultrapure water. The PCR amplification program was set as follows: pre-denaturation at 94℃ for 3~5 min; 35 cycles of amplification: denaturation at 94℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 35 s, final extension at 72℃ for 10 min, and storage at 4℃. The specific primer sequences, corresponding chromosomes, and amplified fragment lengths are shown in the table below.

[0012]

[0013] The 16 genotyping markers include 8 common variant markers, 6 rare variant markers, and 2 uncommon variant markers. The common variant markers have a variation rate of 45-55% in 134 garlic core germplasm accessions and are evenly distributed across the 8 garlic chromosomes. The rare variant markers have a variation rate of about 10%, and the uncommon variant markers have a variation rate of less than 5%. Both types of markers are also evenly distributed across the 8 chromosomes. Two functional markers for alliin content were designed using primers targeting an 82 bp insertion / deletion variant (DEL82) in intron 5 of the AsGGT2 gene and an 851 bp insertion / deletion variant (INS851) in exon 4 of the AsFMO11 gene. The forward primer for the AsGGT2 marker was located in the 82 bp deletion region, while the reverse primer spanned the intron splicing site and extended to exon 6. The primer for the AsFMO11 marker was located within the 851 bp insertion fragment. An internal control marker was designed targeting the conserved plant gene phytopenic oleoresin dehydrogenase (PDS), with an amplified fragment length of 286 bp, used to specifically determine the integrity and validity of the DNA sample. Molecular identification of multiple known garlic varieties was obtained through large-scale sampling in the core areas of their varietal origin. These varieties include Jinxiang white garlic, space garlic, Pizhou white garlic, Cangshan four-six clove garlic, Qixian white garlic, Ershui early garlic, Zhengyue early garlic, Daqingke garlic, Cangshan garlic, Yunnan purple garlic, and Jinxiang purple garlic.

[0014] The present invention also includes a method for accurate identification of garlic varieties, which uses the construction method described in the present invention to obtain a garlic variety fingerprint spectrum.

[0015] This precise identification method includes the following steps:

[0016] (1) Sample DNA extraction: Leaf tissue samples of garlic to be tested were collected, and genomic DNA was extracted using the CTAB method;

[0017] (2) PCR amplification: The genomic DNA was amplified by PCR using a specific primer combination, which corresponds to 16 genotyping markers, 2 allicin content functional markers and 1 internal reference marker;

[0018] (3) DNA quality assessment: DNA sample quality is assessed by the internal control label band. If the internal control label has no clear band, the DNA sample is determined to have degraded or failed to be extracted, and the identification is terminated. If the internal control label band is clear, the DNA sample is determined to be qualified.

[0019] (4) Genotype reading and fingerprint pattern construction: For qualified DNA samples, the bands of 16 genotyping markers and 2 functional markers are read sequentially. The amplification product with the corresponding band is counted as "1" and the absence of the corresponding band is counted as "0", thus forming the molecular fingerprint pattern of the variety to be tested;

[0020] (5) Variety identification: The molecular fingerprint spectrum of the variety to be tested is compared with the garlic variety fingerprint spectrum obtained by the construction method described in this invention to complete the variety tracing and identification.

[0021] This invention can also preliminarily determine the alliin content level of the tested variety based on the genotypes of two alliin functional markers.

[0022] The garlic variety fingerprint obtained by the construction method described in this invention can be used for garlic variety identification, garlic genome structural variation identification, or alliin synthesis gene identification in garlic.

[0023] This invention utilizes de novo assembly and pan-genome construction of the garlic genome to identify numerous genetically stable large-scale structural variation sites; it identifies functional structural variations in the key alliin synthesis genes AsGGT2 and AsFMO11; and further constructs a fingerprinting system containing 16 genotyping markers and 2 alliin functional markers, establishing a standardized variety identification process. This method requires only agarose gel electrophoresis for detection, is simple to operate, low in cost, and provides stable results. It can accurately identify garlic varieties and indirectly assess the content of the core flavor trait, alliin, providing reliable technical support for garlic germplasm resource protection, new variety rights protection, and standardized management of market varieties.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0025] (1) High technical stability: The structural variation markers used in this invention have significantly better genetic stability than traditional markers such as SSR and SNP, and the detection results are not affected by laboratory or batch differences, and the data can be standardized and shared.

[0026] (2) Low operating cost: The detection can be completed with just a regular PCR instrument and agarose gel electrophoresis. No expensive equipment is required, making it suitable for use by grassroots agricultural technology extension departments and seed companies.

[0027] (3) Functional integration: It integrates variety classification markers and quality function markers, and can simultaneously realize accurate identification of garlic varieties and preliminary assessment of alliin content, greatly enhancing the industrial application value of the technology.

[0028] (4) Solving industry pain points: By establishing molecular identity cards for the main garlic varieties, the problem of variety mixing of “different names for the same species and the same name for different species” can be effectively solved, providing a basis for variety rights protection and market supervision. Attached Figure Description

[0029] Figure 1 The image shows the identification information of garlic structural variation in Example 1, where a is the phylogenetic tree position of the five sequenced varieties and their bulb appearance, b is the alliin content of the bulbs of the five sequenced varieties, c is the SV length distribution, and d is the overlap ratio of SV removal and repetitive sequence positions.

[0030] Figure 2 The images show the alliin synthesis genes AsGGT2 and AsFMO11 in Example 1. Image a compares the alliin content in plants overexpressing AsGGT2 and AsFMO11. Image b is an IGV diagram showing the 82 bp deletion variant (DEL82) in the AsGGT2 intron. Image c is a slightly different IGV diagram showing the exon insertion variant (INS851) in AsFMO11. Image d shows the 134 core germplasms divided into three groups based on the DEL82 (left image) and INS851 genotypes, and the alliin content in each group is compared.

[0031] Figure 3 These are gel images of the 11 main garlic varieties in Example 1;

[0032] Figure 4 This is a fingerprint image of 11 types of garlic from Example 1;

[0033] Figure 5 This is a gel image used for identifying the garlic variety in Example 2. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0035] Example 1

[0036] 1. Construction of molecular identity cards for 11 major garlic varieties

[0037] Eleven major garlic varieties cultivated nationwide were selected, including Jinxiang white garlic, space garlic, Pizhou white garlic, Cangshan four-six clove garlic, Qixian white garlic, Ershui early garlic, Zhengyue early garlic, Daqingke garlic, Cangshan garlic, Yunnan purple garlic, and Jinxiang purple garlic. Fingerprint mapping was completed, and the specific steps are as follows:

[0038] 1.1 Sampling in the Core Area: To ensure the uniformity and representativeness of the sampling, the standardized sampling specifications for variety identification were strictly followed. Sampling was conducted in different ecological planting areas in Jintang County (Ershui Early) in Sichuan Province, Pengzhou City (Zhengyue Early) in Sichuan Province, Jinxiang County (Space Garlic, Jinxiang White Garlic, Jinxiang Purple Garlic) in Shandong Province, Lanling County (Cangshan Four-Six Cloves, Cangshan Garlic) in Shandong Province, Shanghe County (Large Highland Barley) in Shandong Province, Qi County (Qixian White Garlic) in Henan Province, Pizhou City (Pizhou White Garlic) in Jiangsu Province, and Eryuan County (Yunnan Purple Garlic) in Yunnan Province. Within each ecological area, uniform cultivation and management conditions were implemented. Random field sampling was conducted, and healthy garlic plants with uniform growth, no pests or diseases, no mechanical damage, and typical agronomical traits were strictly selected. For each garlic variety tested, three healthy, independently growing individual plants were randomly selected from each ecological area, maintaining a reasonable field spacing of ≥50 cm between plants to avoid overlapping kinship and nutrient competition interference, ensuring the independence and representativeness of the sampling. During sampling, the outer old leaves of the garlic were carefully peeled off, and the tender new heart leaves inside the plant were precisely cut. Heart leaf samples from three individual plants of the same variety were quickly collected and mixed in equal volumes. Dirt and other impurities on the leaf surface were manually removed, and water stains were gently wiped off with sterile, dust-free filter paper to complete sample purification. The processed fresh leaf samples were quickly cut into small pieces, immediately placed into sterile cryovials, and sealed. The exposure time at room temperature was minimized throughout the process. The cryovials containing the samples were rapidly immersed in liquid nitrogen for at least 30 minutes to fully inhibit nuclease activity and prevent leaf tissue metabolism and DNA degradation. After rapid freezing, all samples were transferred to an ultra-low temperature freezer at -80℃ for long-term storage in the dark, for subsequent genomic DNA extraction. This minimizes the interference of environmental factors on molecular detection and ensures accurate and reliable test results.

[0039] 1.2 Molecular Identification Construction Process: The experiment was conducted according to the standard procedure for molecular identification of garlic varieties. High-quality genomic DNA was extracted from the leaves of the tested garlic, and the DNA concentration and purity were tested to meet the requirements for molecular marker amplification. The detailed process is as follows:

[0040] Genomic DNA was extracted using the CTAB method, with samples taken from leaves. DNA concentration was measured, and agarose gel electrophoresis was performed to verify the absence of degradation and protein contamination. The DNA was then uniformly diluted to 20–50 ng / μL and stored at -20 °C.

[0041] This invention combines common variant markers, rare variant markers, uncommon variants, and functional markers to construct a fingerprint identification system containing 16 molecular markers. Furthermore, this invention also designs an internal control marker to assess the quality of the DNA sample being tested. Details are as follows:

[0042] (1) Common variation markers. Variations with a variation rate of 45-55% were screened from 134 core germplasms (M2, M4, M5, M8, M9, M12, M13, and M16, i.e., wild-type alleles and mutant genotypes each accounted for about half). One variation was selected from each chromosome to design primers. Garlic has 8 chromosomes, so a total of 8 pairs of primers were designed (see Table 1). Because of their high variation rate in the germplasm, the combination of 8 primer pairs can serve as the basic framework for fingerprinting, which can basically complete the task of variety identification.

[0043] (2) Rare and infrequent variant markers. Six rare variants (M1, M3, M7, M10, M11, and M15, with a variation rate of ~10% in 134 core germplasms) and two infrequent variants (M6 and M14, with a variation rate of less than 5% in 134 core germplasms) were selected. These eight variants are evenly distributed across eight chromosomes, and primers were designed (see Table 1). Since these variants only appear in a few varieties, their combination can further improve the accuracy of variety identification.

[0044] (3) Functional markers for allicin content (M17, M18). For the deletion variant of AsGGT2, a front primer was designed located in the 82bp deletion variant region, and a back primer was designed located in the 6th exon region, crossing the splice site sequence in between. Figure 2 (b). For AsFMO11, primers were designed within its 851 bp region (see Table 1). These two variations are directly related to the accumulation of alliin in garlic bulbs ( Figure 2 (d) can be used as a functional marker to preliminarily evaluate the allicin content of a variety.

[0045] (4) Internal control marker. 18S is a conserved gene in plants, and its deletion will lead to plant albinism. 18S gene primers were designed as internal control markers (Table 1) to determine the DNA quality of the test samples.

[0046] Table 1. Molecular marker information used to construct garlic fingerprint profiles

[0047]

[0048] (5) PCR amplification was performed on 11 tested garlic varieties. The PCR reaction system consisted of 10 μL standard: 5.0 μL of 2×TaqPCR Master Mix; 0.5 μL of upstream primer (10 μmol / L); 0.5 μL of downstream primer (10 μmol / L); 1.0 μL of template DNA (20~50 ng / μL); and 3.0 μL of sterile ultrapure water. The PCR amplification program was set as follows: pre-denaturation at 94℃ for 3~5 min; cyclic amplification (35 cycles): denaturation at 94℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 35 s, final extension at 72℃ for 10 min, and storage at 4℃. For agarose gel electrophoresis, a 3% agarose gel was prepared, melted, and nucleic acid dye was added. The gel was then poured, combed, and allowed to solidify at room temperature. Add 5 μL of PCR product to each well, add DNA marker and 1×TAE buffer to both wells, maintain a constant voltage of 150 V, and electrophoresis for 20 min until the bands are clearly separated. Take a picture under UV light using a gel imaging system, and statistically analyze the band classification results. Record the presence of a band as 1 and the absence of a band as 0. The gel image is shown below. Figure 3 As shown;

[0049] (6) Integrate the labeled typing data, perform coding visualization, and construct fingerprint maps.

[0050] according to Figure 3 Establish standardized molecular identity cards based on gel imaging results, such as... Figure 4 As shown, the constructed molecular identity card features are clear and highly recognizable, serving as a reference benchmark. This effectively solves problems such as homonymous varieties, synonyms, and variety confusion in the garlic market, providing technical support for garlic germplasm resource identification, variety authenticity verification, seedling purity testing, and germplasm traceability.

[0051] 1.3 Determination of alliin content in bulbs of 11 major garlic varieties

[0052] Accurately weigh 25 g (accurate to 0.1 mg) of peeled fresh garlic. Inactivate the enzyme in a microwave oven on high for 2 min. Place the garlic in a homogenizer, add 50 mL of water, and homogenize for 2 min. Quantitatively transfer the homogenate to a 100 mL volumetric flask, add water to the mark, shake well, and sonicate for 30 min. Centrifuge for 15 min (8000 r / min). Accurately transfer 1 mL of the supernatant to a 50 mL volumetric flask, dilute to the mark with the mobile phase, and shake well. Determine the alliin content in the extract using high-performance liquid chromatography (HPLC, Shimadzu). Filter the extract through a 0.22 μm microporous membrane before chromatographic analysis. The HPLC conditions are as follows: column: C18 (4.6 × 250 mm, 5 µm); mobile phase: 0.06% trifluoroacetic acid aqueous solution; flow rate: 0.5 mL / min; column temperature: 30 ℃; detection wavelength: 214 nm; injection volume: 10 µL.

[0053] Allicin standard stock solution (200 mg / L): Weigh 0.020 g (accurate to 0.00001 g) of allicin standard substance into a 100 mL volumetric flask, dissolve in water, dilute to the mark, shake well, store at 4℃, shelf life 1 month.

[0054] Preparation of standard curves: Accurately pipette a certain amount of allicin standard stock solution and prepare a series of standard solutions with concentrations of approximately 1 mg / L, 5 mg / L, 10 mg / L, 25 mg / L, 50 mg / L, and 100 mg / L using water for determination by high performance liquid chromatography.

[0055] Take the test solution and the corresponding concentration of standard solution and determine them according to the chromatographic reference conditions. Perform single-point or multi-point calibration, use retention time for qualitative analysis, and use the external standard method for quantification using chromatographic peak area. The response values ​​of allicin in the test solution and standard working solution should be within the linear range of the instrument. The mass fraction of allicin in garlic is calculated according to the following formula:

[0056]

[0057] In the formula: ω—the mass fraction of alliin in garlic, expressed as %

[0058] D—Dilution factor of the sample;

[0059] Ax — Peak area of ​​allicin in the sample solution;

[0060] CS – Concentration of the standard solution, expressed in milligrams per milliliter (mg / mL).

[0061] AS—Peak area of ​​allicin in standard solution;

[0062] W—Sample size, in grams (g);

[0063] The measurement results are expressed as the arithmetic mean of parallel measurements, and the calculation results are retained to 3 significant figures.

[0064] Example 2: Precise Identification of Major Garlic Varieties

[0065] Sample collection: Young leaves of three garlic varieties (numbered W1-W3) labeled "Jinxiang White Garlic" were collected from the market, with three replicates for each variety.

[0066] DNA extraction and PCR amplification: DNA was extracted using the CTAB method and PCR amplification was performed according to the primers in Table 1 and the procedure in Example 1.

[0067] Electrophoresis and genotyping: The marker band readings for W1 and W3 were 100010001011101011, completely consistent with the molecular identification of Jinxiang white-skinned garlic in Table 2. Furthermore, both M17 and M18 markers were mutants, indicating low alliin content. Subsequent HPLC analysis showed alliin content of 0.81 mg / g and 0.75 mg / g for W1 and W3 respectively, consistent with the initial determination, confirming that these two varieties are indeed Jinxiang white-skinned garlic.

[0068] The marker band reading for W2 was 110110001011101011, which is completely inconsistent with the molecular identification of Jinxiang white-skinned garlic in Table 2. Therefore, this variety is not Jinxiang white-skinned garlic. Markers M17 and M18 are both mutants, indicating low alliin content. Subsequent simplified genome sequencing confirmed that W2 is definitely not Jinxiang white-skinned garlic. HPLC analysis showed an alliin content of 0.70 mg / g, consistent with the initial assessment (see...). Figure 5 ).

[0069] Example 3: Identification of structural variations in the garlic genome

[0070] Garlic populations can be divided into 5 groups. In this study, four garlic materials and the Ershui Early variety (with published genome assembly) were selected as representatives of each group for genome comparison. These materials showed significant differences in bulb morphology and alliin content. Figure 1 (a) and (b) , de novo genome assembly was performed on four garlic samples. Using an average of approximately 17× PacBio HiFi sequencing data, initial contig sequences of 16.84–17.07 Gb were obtained, with N50 values ​​of 2.08–12.88 Mb. The integrity of the four assembled genomes was verified by evaluating the HiFi read length ratio (98.64%–99.57%) and genome coverage (98.27%–99.05%) aligned to the assembled sequences, combined with a BUSCO universal single-copy homolog recovery rate of 95.2%–96.1%. Furthermore, the assembly quality score, assessed using a reference-free kmer analysis method, ranged from 42.65 to 46.09. In summary, these results demonstrate excellent accuracy, integrity, and continuity in the assembly of these four garlic genomes.

[0071] Next, the four newly assembled genomes were integrated with two published garlic genomes to construct a garlic pan-genome map. The final pan-genome map contained 1,395,397 large-scale structural variants (≥50 bp in length), including 570,729 insertions and 824,668 deletions. Subsequently, PanGenie was used to genotype the resequencing datasets of 134 core garlic germplasms, identifying 1,009,266 structural variants (72.33%) in the population, indicating that these structural variants were reliable and representative, collectively constituting the graph-based garlic pan-genome. Among the successfully genotyped structural variants, approximately 38.39% were longer than 2 kb (…). Figure 1 (c). Furthermore, it was found that 88.67% of the structural variations in the 134 samples were located in repetitive regions of the garlic genome, and the proportion of structural variations located in repetitive regions ranged from 89.90% to 94.28% across the five taxa. Figure 1 (d) indicates that transposon evolution is an important factor driving structural variations in the garlic genome.

[0072] 2. Identification of the allicin synthesis gene

[0073] γ-Glutamyltransferase (GGT) and flavin monooxygenase (FMO) are two key enzymes catalyzing the synthesis of alliin. GGT removes the glutamyl group from γ-glutamylallyl cysteine ​​to obtain allyl cysteine, while FMO catalyzes the sulfur oxidation of allyl cysteine ​​to form alliin. Analysis of exogenously applied alliin revealed differential expression between the garlic GGT gene AsGGT2 and the FMO gene AsFMO11. Therefore, overexpression vectors for both genes were constructed and overexpressed in garlic using *Agrobacterium rhizogenes*. Liquid chromatography analysis of the alliin content in transgenic garlic showed that their overexpression significantly led to alliin accumulation, confirming their involvement in alliin biosynthesis. Figure 2 (a) It is worth noting that the fifth intron of AsGGT2 contains an 82 bp insertion / deletion variant (DEL82), which contains an intron splicing site. This deletion variant causes a splicing error in the coding sequence. Figure 2 (b). Furthermore, an 851 bp insertion (INS851) exists in exon 4 of AsFMO11, which alters the sequence of the encoded protein ( Figure 2 (c). Among 134 core garlic germplasms, those containing these two structural variations had significantly lower alliin content in their bulbs than those without the variations. Figure 2 (d). The results indicate that these two variants, by altering the corresponding protein sequences, have a significant impact on the accumulation of allicin.

Claims

1. A method for constructing a fingerprint spectrum of a garlic variety, characterized in that, Includes the following steps: (1) Sample DNA extraction: Leaf tissue samples of several known garlic varieties were collected, and genomic DNA was extracted using the CTAB method; (2) PCR amplification: Genomic DNA was amplified by PCR using a specific primer combination. The specific primer combination corresponded to 16 genotyping markers, 2 allicin content functional markers and 1 internal reference marker. (3) DNA quality assessment: DNA sample quality is assessed by the internal control label band. If the internal control label has no clear band, the DNA sample is considered to have degraded or failed to be extracted; if the internal control label band is clear, the DNA sample is considered to be qualified. (4) Genotype reading and fingerprint pattern construction: For qualified DNA samples, the bands of 16 genotyping markers and 2 functional markers are read in sequence. The amplification product with the corresponding band is counted as "1" and the absence of the corresponding band is counted as "0", thus forming the molecular fingerprint patterns of multiple garlic varieties.

2. The construction method according to claim 1, characterized in that, In step (2), the PCR amplification reaction system consists of 10 μL standard, 5.0 μL of 2×Taq PCR Master Mix, 0.5 μL of upstream primer (10 μmol / L) for each label, 0.5 μL of downstream primer (10 μmol / L) for each label, 1.0 μL of template DNA (20~50 ng / μL), and 3.0 μL of sterile ultrapure water.

3. The construction method according to claim 1, characterized in that, In step (2), the PCR amplification program is set as follows: pre-denaturation at 94℃ for 3-5 min; 35 cycles of amplification: denaturation at 94℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 35 s, final extension at 72℃ for 10 min, and storage at 4℃.

4. The construction method according to claim 1, characterized in that, In step (2), the specific primer sequences, corresponding chromosomes, and amplified fragment lengths are shown in the table below:

5. The construction method according to claim 4, characterized in that, The 16 genotyping markers include 8 common variant markers, 6 rare variant markers, and 2 uncommon variant markers. The common variant markers have a variation rate of 45-55% in 134 garlic core germplasm accessions and are evenly distributed across the 8 garlic chromosomes. The rare variant markers have a variation rate of about 10%, and the uncommon variant markers have a variation rate of less than 5%. Both types of markers are also evenly distributed across the 8 chromosomes.

6. The construction method according to claim 4, characterized in that, Two functional markers for allicin content were primers designed for the 82 bp insertion / deletion variant (DEL82) in the 5th intron of the AsGGT2 gene and the 851 bp insertion / deletion variant (INS851) in the 4th exon of the AsFMO11 gene. The forward primer for the AsGGT2 functional marker is located in the 82 bp deletion region, and the reverse primer spans the intron splicing site and extends to exon 6. The AsFMO11 functional marker primer is located inside the 851 bp insertion fragment. The internal control marker is a primer designed for the plant conserved gene phytopene dehydrogenase (PDS), and its amplified fragment length is 286 bp, which is used to specifically determine the integrity and validity of the DNA sample.

7. The construction method according to claim 1, characterized in that, The molecular identification of several known garlic varieties was formed through large-scale sampling in the core areas of their place of origin. These known garlic varieties include Jinxiang white garlic, space garlic, Pizhou white garlic, Cangshan four-six clove garlic, Qixian white garlic, Ershui early garlic, Zhengyue early garlic, Daqingke garlic, Cangshan garlic, Yunnan purple garlic, and Jinxiang purple garlic.

8. A precise identification method for garlic varieties, characterized in that, The garlic variety fingerprint obtained using the construction method according to any one of claims 1-7 includes the following steps: (1) Sample DNA extraction: Leaf tissue samples of garlic to be tested were collected, and genomic DNA was extracted using the CTAB method; (2) PCR amplification: The genomic DNA was amplified by PCR using a specific primer combination, which corresponds to 16 genotyping markers, 2 allicin content functional markers and 1 internal reference marker; (3) DNA quality assessment: DNA sample quality is assessed by the internal control label band. If the internal control label has no clear band, the DNA sample is determined to have degraded or failed to be extracted, and the identification is terminated. If the internal control label band is clear, the DNA sample is determined to be qualified. (4) Genotyping and fingerprinting: For qualified DNA samples, the bands of 16 genotyping markers and 2 functional markers are read sequentially. The amplification product with a corresponding band is counted as "1" and the absence of a corresponding band is counted as "0", thus forming the molecular fingerprint of the variety to be tested; (5) Variety identification: The molecular fingerprint spectrum of the variety to be tested is compared with the garlic variety fingerprint spectrum obtained by the construction method described in any one of claims 1-6 to complete the variety tracing and identification.

9. The precise identification method according to claim 8, characterized in that, The alliin content level of the tested variety can also be preliminarily determined based on the genotypes of the two alliin functional markers.

10. The garlic variety fingerprint obtained by the construction method according to any one of claims 1-7 can be used for garlic variety identification, garlic genome structural variation identification, or alliin synthesis gene identification in garlic.

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