SSR (Simple Sequence Repeat) molecular marker primer related to dormancy of Chinese chives as well as detection method and application of SSR molecular marker primer

Abstract

The invention discloses an SSR molecular marker primer related to leek dormancy as well as a detection method and application of the SSR molecular marker primer. Aiming at the problems that the traditional dormancy character identification of Chinese chives depends on field morphological observation as long as 4 months, the efficiency is low and special molecular markers are lacked, seven pairs of specific SSR primers (SSR005 / 170 / 249 / 325 / 423 / 478 / 610) are developed based on cross-species genome re-sequencing (taking green Chinese onion as a reference genome, and the comparison rate is 18.76%-18.89%) for the first time. The method comprises the following steps: establishing a matched detection system: rapidly extracting DNA (deoxyribonucleic acid) by a CTAB (cetyltrimethyl ammonium bromide) method, optimizing a PCR (polymerase chain reaction) system (20 mu L of DNA containing a template of 30 ng, 0.4 U of Taq enzyme, 0.2 mu M of primer, 2.0 mM of Mg < 2 + >, 0.2 mM of dNTPs, and annealing at 57 DEG C), and performing 8% polyacrylamide gel electrophoresis combined with silver staining development, thereby accurately identifying the dormancy character within 7 days. For example, the SSR423 primer has non-differentiated non-dormancy / dormancy germplasm at 177bp, and the coincidence rate of the detection result of 52 germplasm (such as dormancy germplasm 21-1 and non-dormancy germplasm 22-7) and the field phenotype reaches 100%.

Description

Technical Field

[0001] The invention provides an SSR molecular marker primer related to leek dormancy, and particularly relates to an SSR molecular marker primer related to leek dormancy, a detection method and an application thereof. Background Art

[0002] Chives are a perennial root vegetable in the genus Allium, part of the Liliaceae family. Native to my country, they are one of the most widely cultivated vegetables in the country, with a global reputation for both variety resources and production technology. As a leafy green vegetable, they offer significant economic benefits, with per-acre income exceeding 30,000 yuan.

[0003] Dormancy is a crucial trait affecting year-round leek production in regions north of the Yellow River. However, leek breeders have not yet prioritized this trait in new cultivar selection. As a result, despite the annual expansion of greenhouse leek cultivation in northern China, market supply continues to plummet from November to the end of February. For many years, leek prices have remained high during these four months, with leeks becoming increasingly scarce during New Year's Day and Spring Festival. Only by prioritizing dormancy as a key breeding target can new leek varieties suitable for year-round production be developed, thereby ensuring a balanced year-round leek supply.

[0004] In order to breed new varieties that can be used for year-round production, it is necessary to develop a method for quickly and accurately identifying the dormancy of leek germplasm. Previous researchers have used morphological indicators in the field or in artificial climate chambers to identify dormancy. This method is inefficient and time-consuming, which seriously restricts the breeding process of new varieties that can be used for year-round production. Molecular marker technology can be used to quickly and accurately evaluate dormancy traits, so it is particularly necessary to develop molecular markers that are closely related to dormancy. Based on the actual needs of leek production, this project develops molecular markers such as SSR and KASP that are closely related to dormancy, establishes a dormancy molecular marker evaluation technology system, uses this system to identify the dormancy traits of germplasm resources and hybrid offspring, and breeds new varieties with different dormancy types for use in year-round leek production. This invention has important application value. In addition, the molecular marker-assisted breeding technology established by the present invention can be used for the study of other traits of leek, which has very important theoretical significance for occupying the international research heights of leek molecular breeding. Summary of the Invention

[0005] In order to solve the above problems, the present application provides an SSR molecular marker primer related to the dormancy of leek and its detection method and application, thereby solving the problem that traditional identification relies on field observations that take 4 months and lacks leek-specific molecular markers, and can accurately distinguish dormancy traits (such as the presence or absence of the 177bp band of SSR423) within 7 days, promoting the selection and breeding of new varieties for year-round production.

[0006] In order to solve the above technical problems, the present invention provides the following technical solution: an SSR molecular marker primer related to leek dormancy, wherein the primer is selected from any one of the following primer combinations:

[0007] (1) SSR005: the upstream primer sequence is TTTTCTTTTTggAgAAAgTgTC, and the downstream primer sequence is CTTgTATCAgCCTTCTTCTTTg;

[0008] (2) SSR170: the upstream primer sequence is CgATTCAAAAACTCAAATAAgC, and the downstream primer sequence is TCTTgTAAgAAACgATCCAATC;

[0009] (3) SSR249: the upstream primer sequence is TTTgCCTAAAgCTTgTAATgTT, and the downstream primer sequence is ACAACACCACATTACACTgAgA;

[0010] (4) SSR325: the upstream primer sequence is ACCAACAAATCAACTCAAAgAg, and the downstream primer sequence is gTCCAAgCTCATCTgTTACATT;

[0011] (5) SSR423: upstream primer sequence is TTgAAgCATCCCATAATTAAAC, downstream primer sequence is CAAATCATCACTACTAACgggT;

[0012] (6) SSR478: upstream primer sequence is TAAAAATCgCATCTTTATCCTg, downstream primer sequence is AAggTTgTgAggTgAgTgTTAg;

[0013] (7) SSR610: The upstream primer sequence is ACACTTgAgAgTggAAACAgA, and the downstream primer sequence is CgTAggCTATgACgAAgAATAg.

[0014] A method for detecting dormancy of leek based on the SSR molecular marker primers according to claim 1, comprising the following steps:

[0015] (1) extracting genomic DNA of the leek sample to be tested;

[0016] (2) using the DNA obtained in step (1) as a template and performing PCR amplification using any primer pair in claim 1;

[0017] (3) Separate and develop the amplified products by electrophoresis, and determine the dormancy trait based on the size or presence of the bands:

[0018] When using SSR423 primers, the presence of a band at 177 bp indicates non-dormant germplasm, while the absence of a band indicates dormant germplasm;

[0019] When using the SSR005 primer, the 235 bp band corresponds to the nondormant germplasm, and the 265 bp band corresponds to the dormant germplasm;

[0020] When using SSR478 primers, the 189 bp band corresponds to nondormant accessions, and the 230 bp band corresponds to dormant accessions;

[0021] When using SSR170, SSR249, SSR325, or SSR610 primers, the dormancy type was determined based on the presence or absence of differential band positions.

[0022] Preferably, the DNA extraction in step (1) adopts the CTAB method, which specifically includes:

[0023] Fresh leaves were added to CTAB buffer and ground in a 65°C water bath for 1 hour;

[0024] Extraction was performed with chloroform:isoamyl alcohol (24:1) and DNA was precipitated with isopropanol;

[0025] After washing with 70% ethanol, the cells were dissolved in TE buffer containing RNase A.

[0026] Preferably, the PCR reaction system in step (2) is 20 μL:

[0027] Template DNA 30ng, Taq enzyme 0.4U, primers 0.2μM each, Mg 2+ 2.0mM, dNTPs0.2mM, 1× PCR buffer;

[0028] Reaction procedure: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 45 s, 35 cycles; final extension at 72°C for 7 min.

[0029] Preferably, in step (3), the amplified products are separated by 8% polyacrylamide gel electrophoresis, and then developed by silver staining after 160V constant voltage electrophoresis.

[0030] The invention discloses an application of SSR molecular marker primers related to leek dormancy. The application of the SSR molecular marker primers in leek molecular marker-assisted breeding includes screening for dormancy traits of individual plants in a hybrid F2 population.

[0031] Preferably: the use of the SSR molecular marker primers in the identification of dormancy traits of leek germplasm resources.

[0032] Preferably: 52 leek germplasm resources were detected using SSR423 primers, among which 21-1, 21-3 to 21-5, 21-7, 21-9, 21-10, 21-12, 21-13, 21-15 to 21-25, 21-30, and 22-15 were dormant germplasms, and the rest were non-dormant germplasms.

[0033] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0034] The present invention solves the problem of low efficiency and long cycle in the prior art of relying on field morphological observation or artificial climate chamber to identify the dormancy of leek. By first developing 7 SSR molecular marker primers (SSR005, SSR170, SSR249, SSR325, SSR423, SSR478, SSR610) closely linked to the dormancy trait of leek and establishing a matching molecular evaluation technology system, the invention uses the leek DNA rapid extraction technology (CTAB method), the optimized SSR-PCR reaction system (20 μL containing 30 ng template DNA, 0.4 U Taq enzyme, 0.2 μM primers, MgCl2, 0.5 μg / mL MgCl2, 0.8 μg / mL MgCl2, 0.9 ... 2+ The high-throughput, rapid, and accurate identification of dormancy traits in leek germplasm was achieved by combining the primers 2.0mM, dNTPs 0.2mM) and procedures (annealing at 57°C), as well as polyacrylamide gel electrophoresis combined with silver staining technology. For example, the SSR423 primer band at 177bp could not distinguish between non-dormant and dormant germplasm, significantly shortening the breeding cycle and overcoming the bottleneck of traditional methods that restrict the selection of new varieties for year-round production.

[0035] Other advantages, objects and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art based on an examination of the following or may be learned from the practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the verification of the SSR molecular marker primer related to the dormancy of leek and its detection method and application SSR005 in the non-dormant pool, dormant pool and some pool-established individual plants; Note: from left to right, the materials are the non-dormant pool, dormant pool and 12 pool-established individual plants.

[0037] Figure 2 This is the verification of the SSR molecular marker primer related to the dormancy of leek and its detection method and application SSR170 in the non-dormant pool, dormant pool and some pool-established individual plants; Note: from left to right, the materials are the non-dormant pool, dormant pool and 10 pool-established individual plants.

[0038] Figure 3This is the verification of the SSR molecular marker primer related to the dormancy of leek and its detection method and application SSR249 in the non-dormant pool, dormant pool and some established pool individual plants; Note: from left to right, the materials are the non-dormant pool, dormant pool and 16 established pool individual plants.

[0039] Figure 4 This is the verification of the SSR molecular marker primer related to the dormancy of leek and its detection method and the applied SSR325 in the non-dormant pool, dormant pool and some established pool individual plants; Note: from left to right, the materials are the dormant pool, the non-dormant pool and 16 established pool individual plants.

[0040] Figure 5 This is the verification of the SSR molecular marker primer related to the dormancy of leek and its detection method and the applied SSR423 in the non-dormant pool, dormant pool and some established pool individual plants; Note: from left to right, the materials are the non-dormant pool, dormant pool and 16 established pool individual plants.

[0041] Figure 6 This is the verification of the SSR molecular marker primer related to the dormancy of leek and its detection method and the applied SSR478 in the non-dormant pool, dormant pool and some established pool individual plants; Note: from left to right, the materials are the non-dormant pool, dormant pool and 20 established pool individual plants.

[0042] Figure 7 This is the verification of the SSR molecular marker primers and detection method related to the dormancy of Chinese chives, and the applied SSR610 in a non-dormant pool, a dormant pool, and some established pool plants. Note: From left to right, the materials are a non-dormant pool, a dormant pool, and 20 established pool plants.

[0043] Figure 8 This is a schematic diagram of the dormancy type detection of 52 germplasm resources using SSR molecular marker primers related to leek dormancy and its detection method and application SSR423; Note: From left to right, the material codes are 21-1 to 21-32, 22-1 to 22-20; the bands indicated by the arrows are non-dormant germplasm. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0045] It should be noted that the terms “vertical”, “horizontal”, “up”, “down”, “left”, “right” and similar expressions used in this document are for illustrative purposes only and do not represent the only implementation method.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains; the terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0047] like Figure 1-7 As shown, the present invention has designed a SSR molecular marker primer related to the dormancy of leek, and listed seven primer combinations including SSR005, SSR170, SSR249, SSR325, SSR423, SSR478, and SSR610. Each primer combination contains corresponding upstream primer sequences and downstream primer sequences. These primers play a key role in the detection of leek dormancy. The invention also provides a method for detecting leek dormancy based on the above-mentioned specific primers, including the steps of genomic DNA extraction, PCR amplification, electrophoresis separation and development, and the dormancy characteristics of leek are judged by observing the size or presence of the amplified product bands. Different primers correspond to different band positions and dormancy type judgment criteria.

[0048] In this embodiment, the present invention is further described in conjunction with the claims:

[0049] Right 1 (core marker): provides 7 pairs of specific SSR primers (SSR005 / 170 / 249 / 325 / 423 / 478 / 610), which is the technical basis for solving the problem of "no available dormancy molecular markers for leek".

[0050] Right 2-5 (method system): Based on right 1, a standardized method for the entire process of "DNA extraction → PCR amplification → electrophoresis development → trait interpretation" is constructed to solve the problem of "long traditional identification cycle".

[0051] Right 3: Limit the CTAB method to extract DNA (to ensure high-quality DNA yield from leek materials)

[0052] Right 4: Define orthogonal optimized PCR system (to ensure primer amplification specificity)

[0053] Right 5: Limited polyacrylamide gel electrophoresis + silver staining (to accurately detect differences in small SSR fragments)

[0054] Right 6-7 (Application Scenario): Based on the marking of right 1 and the method of right 2, two major applications are realized:

[0055] Right 6: Rapid identification of germplasm dormancy (instead of field observation)

[0056] Right 7: Clarify the validation data of SSR423 in 52 germplasms (provide typical application cases)

[0057] The core role of solving existing technical problems:

[0058]

[0059] Breakthrough synergy

[0060] Breaking the genomic gap limit:

[0061] The marker development of Quan 1 was based on cross-species genome resequencing (the Welsh onion reference genome), and 9,860 SSR sites were obtained in an internationally free leek genome background (section 3.1.1 of the document), filling the technical gap.

[0062] Achieve "accurate and efficient" identification:

[0063] The combination of methods 2-5 increased the accuracy of identification from 75% of field observation to 100% (Section 5 of the document) and reduced the time from 120 days to 7 days.

[0064] Promoting the industrialization of seed breeding:

[0065] The application verification of Quan 6-7 makes dormancy a quantifiable breeding target, directly supporting the selection and breeding of "new varieties for year-round production" (document background section), and solving the livelihood problem of "it is difficult to get leeks" during New Year's Day / Spring Festival.

[0066] Claims 1-7 form a closed-loop technical solution of "marker development → detection standardization → application implementation". Their synergistic effect completely overcomes the three major bottlenecks of "no marker, long cycle, and low accuracy" in the identification of leek dormancy traits, providing core tools for molecular design breeding of leek.

[0067] Twenty individual plants of dormant and non-dormant leek were extracted, and 10 plants from each pool were pooled in equal amounts to construct two dormant pools and two non-dormant pools. PCR amplification of the four DNA pools was performed using 670 synthetic SSR primer combinations. Polymorphic primers were repeatedly tested, and then PCR amplification and electrophoresis were performed on individual plants from the pools. Seven SSR molecular markers tightly linked to the dormancy trait were ultimately identified. The primer sequences are shown in Table 1.

[0068] like Figure 5-7As shown, this detection method can also be used to derive specific implementation steps, such as the specific operational process for DNA extraction using the CTAB method, the detailed preparation of the PCR reaction system, and the separation of amplified products using 8% polyacrylamide gel electrophoresis. Furthermore, the present invention describes the application of this SSR molecular marker primer in the identification of dormancy traits in leek germplasm resources. By detecting leek germplasm resources using specific primers such as SSR423, dormant and non-dormant germplasm can be distinguished.

[0069] In one or more feasible embodiments, Example 1: Development and verification of SSR molecular marker primers

[0070] (1) Genome resequencing: One dormant and one non-dormant leek accession were selected and whole-genome resequencing was performed using the genomes of closely related species of the Allium genus (welsh onion, onion, garlic, leek) as reference genomes (Shenzhen Chengqi Biotechnology Co., Ltd.). The results showed that the test material had the highest genome alignment rate with the leek genome (18.76% / 18.89%). Based on this, 9860 SSR differential sites were obtained, of which dinucleotide repeats accounted for 51.01% (Table 1).

[0071] (2) Primer development and screening: 1115 pairs of SSR primers were designed using Primer 5.0, and 670 pairs were synthesized. DNA pools were constructed: two dormant pools and two non-dormant pools (each pool was composed of equal amounts of DNA from 10 individual strains of the same type). Using the DNA pools (dormant pools / non-dormant pools) as templates for initial screening, and then verified using individual strains from the pools, seven pairs of SSR primers linked to the dormancy trait were obtained (Table 2).

[0072] (3) Marking verification: Take SSR005 as an example ( Figure 1 ), the non-dormant pool and individual plant (individual plant in pool construction) amplified a 235bp band, and the dormant pool and individual plant (individual plant in pool construction) amplified a 265bp band.

[0073] Example 2: Detection method of leek dormancy

[0074] (1) DNA extraction: Take 4-5 young leek leaves, add 650 μl CTAB buffer, grind, and bathe at 65°C for 1 h; extract with chloroform:isoamyl alcohol (24:1), precipitate with isopropanol, wash with 70% ethanol, and dissolve in TE buffer containing RNase A.

[0075] (2) PCR amplification: Orthogonal optimization system (Table 3) was used: 20 μL containing 30 ng template DNA, 0.4 U Taq enzyme, 0.2 μM primers, Mg 2+ 2.0mM, dNTPs 0.2mM. Program: 94℃ initial denaturation 4min; 35 cycles (94℃ 30s→57℃ 30s→72℃ 45s); 72℃ extension 7min ( Figure 1electrophoresis verification).

[0076] (3) Product detection: 8% polyacrylamide gel (160V) electrophoresis separation, silver staining development: fixative (10% ethanol + 0.5% glacial acetic acid) 5 min → silver nitrate staining solution (3 g / L AgNO3 + 0.15% formaldehyde) 7 min → water washing → developer (1.5% NaOH + 0.2% formaldehyde) 5 min → stop development (same as fixative).

[0077] Example 3: Identification of dormancy traits of germplasm resources

[0078] The SSR423 primer was used to detect 52 leek germplasms (Table 4): the dormant germplasms such as Xiaohonggen (21-1) in Wuan, Hebei and Dulegang Village (21-30) in Kaifeng, Henan had no band at 177bp ( Figure 8 The Baiguoshu Village, Suizhou, Hubei (22-15) is a dormant germplasm; the non-dormant germplasm, such as the Chaqi Town, Huarong, Hunan (22-7), shows a clear band at 177 bp ( Figure 8 The identification results were 100% consistent with the field phenotype.

[0079] It is important to note that, as the leek genome sequence has not yet been released internationally, this study used the genomes of allium, onion, garlic, and leek, all members of the same genus, as reference genomes. Leaf DNA from two leek germplasm accessions with different dormancy traits was extracted and sent to Shenzhen Chengqi Biotechnology Co. for whole-genome resequencing. Comparative analysis revealed that the highest genome alignment rates between the test accessions and the leek genome were 18.76% and 18.89%, respectively, while alignment rates with garlic, onion, and leek were only 6-9%. Using the 18.76% and 18.89% of the leek genome, respectively, bioinformatics analysis revealed 9,860 differentially expressed single-stranded sequence (SSR) sites (Table 1).

[0080] Table 1 Distribution of SSRs in different repeat units

[0081]

[0082] Furthermore, the different bands amplified by the seven SSR primers were not sequenced.

[0083] The band of primer SSR005 in the non-dormant pool and individual plant was approximately 235 bp, and the band in the dormant pool and individual plant was approximately 265 bp.

[0084] The differential band of SSR170 primer appeared at 238bp. There was a band at 238bp in the non-dormant pool and individual plant, but no band at 238bp in the dormant pool and individual plant.

[0085] The differential band of SSR249 primer appeared at 316bp. There was a band at 316bp in the non-dormant pool and individual plant, but no band at 316bp in the dormant pool and individual plant.

[0086] The differential band of SSR325 primer appeared at 260bp. There was a band at 260bp in the dormant pool and individual plant, but no band at 260bp in the non-dormant pool and individual plant.

[0087] The differential band of SSR423 primer appeared at 177bp. There was no band at 177bp in the dormant pool and individual plant, but there was no band at 177bp in the non-dormant pool and individual plant.

[0088] The SSR478 primer produced a band of approximately 189 bp in the non-dormant pool and individual plant, and a band of approximately 230 bp in the dormant pool and individual plant.

[0089] The differential band of SSR610 primer appeared at 235bp. There was no band at 235bp in the non-dormant pool and individual plant, but there was a band at 235bp in the dormant pool and individual plant.

[0090] Specifically, the differential band of SSR423 primers appeared at 177 bp, there was no band at 177 bp in dormant germplasm, and there was a band at 177 bp in non-dormant germplasm.

[0091] Primer 5.0 software was used to design SSR primers from 9860 differential loci, resulting in a total of 1115 SSR primer pairs. 670 primer pairs were synthesized using an established SSR reaction system. DNA from two different dormancy types of leek was used as templates for amplification. Relevant molecular markers were screened in both dormant and non-dormant pools, and molecular marker analysis and validation were performed on individual plants in the established pools. Seven SSR markers closely linked to dormancy were ultimately identified. The primer sequences are shown in Table 2.

[0092] Table 2 Primer sequences of 7 SSR molecular markers closely linked to dormancy traits

[0093]

[0094] For additional information, 52 different types of leek germplasm resources were collected from Hebei, Gansu, Henan, Hunan, Hubei and other places. (Those starting with 21 were collected in 2021, and those starting with 22 were collected in 2022).

[0095] Table 2 Information of collected germplasm resources

[0096]

[0097]

[0098] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An SSR molecular marker primer related to leek dormancy, characterized in that: The primers are selected from any one of the following primer combinations: (1) SSR005: the upstream primer sequence is TTTTCTTTTTggAgAAAgTgTC, and the downstream primer sequence is CTTgTATCAgCCTTCTTCTTTg; (2) SSR170: the upstream primer sequence is CgATTCAAAAACTCAAATAAgC, and the downstream primer sequence is TCTTgTAAgAAACgATCCAATC; (3) SSR249: the upstream primer sequence is TTTgCCTAAAgCTTgTAATgTT, and the downstream primer sequence is ACAACACCACATTACACTgAgA; (4) SSR325: the upstream primer sequence is ACCAACAAATCAACTCAAAgAg, and the downstream primer sequence is gTCCAAgCTCATCTgTTACATT; (5) SSR423: upstream primer sequence is TTgAAgCATCCCATAATTAAAC, downstream primer sequence is CAAATCATCACTACTAACgggT; (6) SSR478: upstream primer sequence is TAAAAATCgCATCTTTATCCTg, downstream primer sequence is AAggTTgTgAggTgAgTgTTAg; (7) SSR610: The upstream primer sequence is ACACTTgAgAgTggAAACAgA, and the downstream primer sequence is CgTAggCTATgACgAAgAATAg.

2. A method for detecting dormancy of leek based on the SSR molecular marker primers according to claim 1, characterized in that: The following steps are involved: (1) extracting genomic DNA of the leek sample to be tested; (2) using the DNA obtained in step (1) as a template and performing PCR amplification using any primer pair in claim 1; (3) Separate and develop the amplified products by electrophoresis, and determine the dormancy trait based on the size or presence of the bands: When using SSR423 primers, the presence of a band at 177 bp indicates non-dormant germplasm, while the absence of a band indicates dormant germplasm; When using the SSR005 primer, the 235 bp band corresponds to the nondormant germplasm, and the 265 bp band corresponds to the dormant germplasm; When using SSR478 primers, the 189 bp band corresponds to nondormant accessions, and the 230 bp band corresponds to dormant accessions; When using SSR170, SSR249, SSR325, or SSR610 primers, the dormancy type was determined based on the presence or absence of differential band positions.

3. The method for detecting SSR molecular marker primers related to leek dormancy according to claim 2, characterized in that: The DNA extraction in step (1) adopts the CTAB method, which specifically includes: Fresh leaves were added to CTAB buffer and ground in a 65°C water bath for 1 hour; Extraction was performed with chloroform:isoamyl alcohol (24:1) and DNA was precipitated with isopropanol; After washing with 70% ethanol, the cells were dissolved in TE buffer containing RNase A.

4. The method for detecting SSR molecular marker primers related to leek dormancy according to claim 2, wherein: The PCR reaction system in step (2) is 20 μL: Template DNA 30ng, Taq enzyme 0.4U, primers 0.2μM each, Mg 2+ 2.0mM, dNTPs0.2mM, 1× PCR buffer; reaction procedure: 94℃ initial denaturation for 4min; 94℃ denaturation for 30s, 57℃ annealing for 30s, 72℃ extension for 45s, 35 cycles; 72℃ final extension for 7min.

5. The method for detecting SSR molecular marker primers related to leek dormancy according to claim 2, characterized in that: In the step (3), the amplified products were separated by 8% polyacrylamide gel electrophoresis, and the electrophoresis was performed at 160V constant voltage and then developed by silver staining.

6. An application of SSR molecular marker primers related to leek dormancy, characterized by: The application of the SSR molecular marker primers in molecular marker-assisted breeding of leek includes application in identification of dormancy traits of germplasm resources.

7. The use of an SSR molecular marker primer related to leek dormancy according to claim 6, characterized in that: SSR423 primers were used to detect 52 leek germplasm resources, among which 21-1, 21-3 to 21-5, 21-7, 21-9, 21-10, 21-12, 21-13, 21-15 to 21-25, 21-30, and 22-15 were dormant germplasm, and the rest were non-dormant germplasm.

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