Chinese chive SSR-PCR reaction technical system and application thereof

Abstract

The invention discloses a leek SSR-PCR reaction system and application thereof, and solves the technical bottlenecks of long period (more than or equal to 4 months), high error rate (more than or equal to 15%) and low flux (less than 10 parts per day) caused by the fact that traditional dormancy character identification depends on field phenotype observation. The system takes a precisely optimized 20 [mu] L reaction system as a core, comprises 30 ng of template DNA, 0.4 U of Taq enzyme, 0.2 [mu] M of primer, 2.0 mM of Mg < 2 + > and 0.2 mM of dNTPs, and realizes stable amplification by combining with a 57 DEG C annealing temperature program; seven SSR molecular markers linked with dormancy characters are synchronously developed, and the SSR423 marker judges dormancy phenotypes through 177bp strip deletion. During application, DNA is extracted through a CTAB method, after amplification of the system, germplasm typing can be completed within 8 hours through polyacrylamide gel electrophoresis detection, 52 germplasm verification shows that the consistency with field phenotypes reaches 100%, and the flux is increased to be larger than 200 parts per day. The technology provides an efficient molecular tool for breeding of leek dormancy-resistant varieties, and annual balanced supply of the market is guaranteed.

Description

Technical Field

[0001] This invention provides a reaction technology system, particularly relating to a chive SSR-PCR reaction technology system and its application. Background Technology

[0002] The "molecular detection technology for dormancy traits in leeks" involved in this application refers to a genotyping system for rapidly identifying dormant phenotypes in leeks. Its core is an SSR-PCR molecular marker technology platform, comprising a DNA extraction module, a PCR amplification module, and an electrophoresis analysis module. This device, by identifying DNA molecular markers (such as SSR sites) linked to dormancy traits, replaces traditional field phenotypic observation, achieving precise and efficient genotyping of germplasm resources.

[0003] Current methods for identifying dormancy traits in chives mainly rely on artificial climate chamber-field observation devices. The basic structure of these devices includes: an environmental simulation unit (artificial climate chamber temperature and light control system); a phenotypic acquisition unit (periodic morphological index recording equipment, such as leaf growth meter); and a data analysis unit (determining dormancy based on the duration of growth stagnation).

[0004] Based on the above system, the following defects and shortcomings exist:

[0005] ①Extremely long cycle: It requires simulating the autumn and winter environment to induce dormancy, and a single test takes ≥4 months, which cannot meet the high-throughput requirements of breeding;

[0006] ② Significant error: Environmental fluctuations (such as temperature deviation ±2℃) lead to misjudgment of morphological indicators;

[0007] ③Low throughput: Relying on manual recording of individual plant phenotypes, the daily average sample processing capacity is <10, while the daily average processing capacity of the device in this application is >200. Summary of the Invention

[0008] To address the aforementioned issues, this application provides a chive SSR-PCR reaction technology system and its application, overcoming the bottlenecks of traditional identification methods, such as long identification cycles (4 months) and high error rates (≥15%). By optimizing components and combining them with the SSR423 marker, it achieves accurate typing within 8 hours, supporting the efficient breeding of chive varieties with different dormancy types.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a leek SSR-PCR reaction technology system, comprising a reaction solution, wherein 20 μL of the reaction solution contains the following components:

[0010] Template DNA 30 ng, Taq DNA polymerase 0.4 U, SSR primer pair 0.2 μM (per primer), Mg 2+ 2.0 mM, 0.2 mM dNTPs, 1×PCR buffer, with the remainder being ddH2O.

[0011] Preferably, the PCR reaction procedure is as follows:

[0012] Pre-denaturation at 94℃ for 4 minutes;

[0013] Denaturation at 94℃ for 30 seconds, annealing at 57℃ for 30 seconds, extension at 72℃ for 45 seconds, for a total of 35 cycles;

[0014] 72℃ for a final extension of 7 minutes;

[0015] Store at 10℃.

[0016] An application of an SSR-PCR reaction technology system for leeks, specifically for detecting dormancy traits in leeks, utilizes SSR molecular marker primer pairs for amplification. These primer pairs are selected from any of the following groups:

[0017] SSR005: Upstream primer TTTTCTTTTTggAgAAAgTgTC (SEQ ID NO:1), downstream primer CTTgTATCAgCCTTCTTCTTTg (SEQ ID NO:2);

[0018] SSR170: Upstream primer CgATTCAAAAACTCAAATAAgC (SEQ ID NO:3), downstream primer TCTTgTAAgAAACgATCCAATC (SEQ ID NO:4);

[0019] SSR249: Upstream primer TTTgCCTAAAgCTTgTAATgTT (SEQ ID NO:5), downstream primer ACAACACCACATTACACTgAgA (SEQ ID NO:6);

[0020] SSR325: Upstream primer ACCAACAAATCAACTCAAAgAg (SEQ ID NO:7), downstream primer gTCCAAgCTCATCTgTTACATT (SEQ ID NO:8);

[0021] SSR423: Upstream primer TTgAAgCATCCCATAATTAAAC (SEQ ID NO:9), downstream primer CAAATCATCACTACTAACgggT (SEQ ID NO:10);

[0022] SSR478: Upstream primer TAAAAATCgCATCTTTATCCTg (SEQ ID NO:11), downstream primer AAggTTgTgAggTgAgTgTTAg (SEQ ID NO:12);

[0023] SSR610: Upstream primer gACACTTgAgAgTggAAACAgA (SEQ ID NO:13), downstream primer CgTAggCTATgACgAAgAATAg (SEQ ID NO:14).

[0024] Preferably, the SSR molecular marker is SSR423, and its amplified fragment size is 177 bp, wherein:

[0025] No 177bp band was found in dormant germplasm;

[0026] A 177bp band was present in the non-dormant germplasm.

[0027] Preferred: Includes the following steps:

[0028] (1) Extract DNA from the leek germplasm to be tested;

[0029] (2) Using the reaction system and procedure described in claims 1-2, PCR amplification of leek germplasm DNA was performed using SSR423 primers;

[0030] (3) Detection of amplification products by electrophoresis:

[0031] If there is no band at 177bp, it is determined to be dormant germplasm; if there is a band at 177bp, it is determined to be non-dormant germplasm.

[0032] A method for identifying dormancy traits in leek germplasm resources includes using the SSR-PCR reaction system described in claims 1-2, performing PCR amplification of the DNA to be tested using SSR423 primers, and distinguishing between dormant and non-dormant phenotypes based on the presence or absence of a 177bp band.

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

[0034] This invention solves the problems of long cycle (requiring 4 months), low efficiency, and susceptibility to environmental interference caused by the reliance on field phenotypic observation for traditional identification of dormancy traits in chives. It develops a precise quantitative SSR-PCR reaction system for chives (containing optimized components: template DNA 30ng, Taq enzyme 0.4U, primers 0.2μM, Mg...). 2+ By using a 2.0mM (2.0mM, dNTPs 0.2mM) and annealing temperature program of 57℃, combined with the first screening of 7 SSR molecular markers linked to dormancy traits (especially the SSR423 marker), rapid genotyping based on the presence or absence of a 177bp band can be achieved. Dormant and non-dormant germplasm can be accurately distinguished in just a few hours, shortening the identification cycle by more than 95%. This provides efficient technical support for the breeding of new varieties of chives with different dormancy types and ensures year-round market supply.

[0035] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0036] Figure 1 The electrophoresis results of the PCR products from an orthogonal experiment of the SSR reaction system of the present invention, which describes a chive SSR-PCR reaction technology system and its application;

[0037] Figure 2 This is an electrophoresis diagram showing the optimal annealing temperature screening for the SSR-PCR reaction technology system for chives and its application according to the present invention.

[0038] Figure 3 This invention provides a chive SSR-PCR reaction technology system and its application process.

[0039] Figure 4 This invention relates to an SSR-PCR reaction technology system for chives and its application in the SSR molecular marker screening process. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] It should be noted that the terms "vertical," "horizontal," "up," "down," "left," "right," and similar expressions used in this article are for illustrative purposes only and do not represent the only possible implementation.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0043] like Figure 1 and Figure 2As shown, a chive SSR-PCR reaction system contains a specific reaction solution, the detailed components of which are as follows: each 20 μL of reaction solution contains 30 ng of template DNA, 0.4 U of Taq DNA polymerase, 0.2 μM of SSR primer pair (each primer), and 2.0 mM of Mg. 2+ The reagents consist of 0.2 mM dNTPs, 1× PCR buffer, and the remainder is made up with ddH2O. The accompanying PCR reaction program is also carefully designed, specifically including: first, a pre-denaturation treatment at 94°C for 4 minutes, followed by 35 cycles, each cycle consisting of denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 45 seconds. After the cycles, a final extension step is performed at 72°C for 7 minutes, and finally, the sample is stored at 10°C.

[0044] In this embodiment, the invention is further described in conjunction with the claims:

[0045] Basic layer (rights 1-2): Establish a standardized SSR-PCR reaction system (rights 1) and a 57℃ annealing procedure (rights 2) to provide a reproducible amplification basis for molecular marker detection.

[0046] Application layer (weights 3-4): Based on the system of weights 1-2, seven dormant trait linked SSR markers are defined (weight 3), and the 177bp band discrimination criterion of SSR423 marker is further focused (weight 4).

[0047] Execution layer (rights 5-6): Based on the technical elements of rights 1-4, construct a complete identification process of "DNA extraction → PCR amplification → electrophoresis interpretation" (right 5), and finally form a standardized detection method for germplasm dormancy traits (right 6).

[0048] Technical Feature Connection Explanation:

[0049] 2.0 mM Mg of weight 1 2+ Together with the annealing temperature of 57°C in Example 2, it ensures amplification specificity (see orthogonal experiment in Example 2);

[0050] The SSR423 primer of right 3 achieved stable amplification with a primer concentration of 0.2 μM in right 1;

[0051] The 177bp stripe interpretation of weight 5 directly references the labeling features of weight 4.

[0052] Furthermore, the optimized system of weights 1-2: by using precise components (such as 30 ng DNA) and an annealing temperature of 57°C (Example 2), non-specific amplification is eliminated, making the electrophoresis results clearly distinguishable (comparison). Figure 2 (The stray bands disappear), avoiding false positive / false negative misjudgments (the misjudgment rate of traditional methods is ≥15% → 0% of this method);

[0053] Step 5-6 process integration: The 4-month field observation (Appendix 1) is compressed into 8 hours of experimental operation (Step 5 (1)-(3)), improving efficiency by 95%.

[0054] Application of markers 3-4: Seven SSR markers (especially SSR423 single marker determination) support simultaneous batch detection (parallel processing of 52 germplasm accessions in Example 4);

[0055] The standardization method of Quan 6 increases the daily processing capacity from <10 samples (traditional manual recording) to >200 samples (based on PCR automation), providing massive sample screening capabilities for breeding.

[0056] The “7-out-of-1” primer set of weight 3 (in Song typeface): When SSR423 becomes ineffective in certain germplasms (e.g., due to mutation), the other 6 marker sets (weight 3) can be switched to ensure universality;

[0057] The basic system of right 1-2: SSR markers that can be transferred to the development of traits such as disease resistance and flavor in leeks (see the end of the background technology section).

[0058] like Figure 3 and Figure 4 As shown, in the practical application of the SSR-PCR reaction technology system for chives, the dormancy trait of chives can be effectively detected by using specific SSR molecular marker primer pairs. The primer pairs involved include SSR005, SSR170, SSR249, SSR325, SSR423, SSR478, and SSR610, each consisting of specific upstream and downstream primers, which play a crucial role in the amplification process. Among them, the SSR423 primer pair is particularly critical, as its amplified fragment size is 177 bp. A 177 bp band is not present in dormant germplasm, but it can be detected in non-dormant germplasm. Using this characteristic, the dormancy trait of leek germplasm resources can be identified through the following steps: First, extract the DNA of the leek germplasm to be tested. Then, use the above-mentioned SSR-PCR reaction system and procedure to perform PCR amplification with SSR423 primers. Finally, detect the amplification product by electrophoresis. If there is no band at 177bp, it is determined to be dormant germplasm; otherwise, it is non-dormant germplasm.

[0059] In this implementation plan,

[0060] In one or more feasible embodiments,

[0061] Example 1: Leek DNA Extraction - Using the small-volume rapid CTAB method described in the supplementary materials:

[0062] Place 4-5 fresh, tender chive leaves (about the size of the centrifuge tube opening) into a 1.5mL centrifuge tube;

[0063] Add 650 μL of CTAB extraction buffer and grind for 3 min until pulverized;

[0064] 65℃ water bath for 1 hour;

[0065] After cooling, add 650 μL of chloroform:isoamyl alcohol (24:1) and mix well for 10 min;

[0066] Centrifuge at 13,000 rpm for 10 min and collect 380 μL of the supernatant;

[0067] Add 700 μL of pre-cooled isopropanol, mix gently, and centrifuge at 13,000 rpm for 10 min.

[0068] Discard the supernatant, wash the precipitate with 100 μL of 70% ethanol, centrifuge and dry;

[0069] Add 100 μL of 0.1×TE (containing 1 μL of RNase A) and incubate at 37°C for 1 h;

[0070] DNA purity (clear bands without degradation) and concentration (adjusted to 30 ng / μL) were determined by 1% agarose gel electrophoresis.

[0071] Example 2: Optimization of SSR-PCR reaction system

[0072] Factor levels (Table 1):

[0073] Template DNA: 30 / 35 / 40 / 45ng

[0074] Taq enzyme: 0.2 / 0.3 / 0.4 / 0.5U

[0075] Primer concentrations: 0.1 / 0.2 / 0.4 / 0.8 μM

[0076] Mg 2+ : 1.0 / 1.5 / 2.0 / 2.5mM

[0077] dNTPs: 0.1 / 0.2 / 0.3 / 0.4mM

[0078] L 16 (4 5 Orthogonal experiment: 16 systems were repeated 3 times, with a reaction volume of 20 μL.

[0079] Table 1 Factors and levels in the SSR reaction system of chives

[0080]

[0081] result( Figure 1 ):

[0082] Optimal combination: template DNA 30 ng, Taq enzyme 0.4 U, primers 0.2 μM, Mg 2+ 2.0 mM, dNTPs 0.2 mM.

[0083] Annealing temperature optimization: the amplification bands were clearest at 57℃. Figure 2 (Verification at a gradient of 50-60℃).

[0084] Example 3: Screening for molecular markers of dormant traits

[0085] DNA pool construction:

[0086] Dormant pool: Mixed DNA from 10 out of 20 dormant germplasms;

[0087] Non-dormant pool: equal amounts of DNA from 10 out of 20 non-dormant germplasms.

[0088] Initial screening by labeling:

[0089] DNA from both pools was amplified using 670 pairs of SSR primers, and polymorphic primers were screened.

[0090] The individual plants in the pond were retested, and seven linkage markers were obtained (Table 2).

[0091] Pond-based single-plant verification:

[0092] Taking SSR423 as an example: the 177bp band appeared in both non-dormant ponds and single plants in ponds, but was absent in both dormant ponds and single plants in ponds.

[0093] Table 2. Characteristics of Linked SSR Marker Amplification

[0094] mark dormant phenotype band characteristics Non-dormant phenotypic band characteristics SSR423 177bp unstriped 177bp with banding SSR005 265bp with banding 235bp with banding SSR170 238bp unstriped 238bp with banding

[0095] Example 4: Identification of dormancy traits in germplasm resources

[0096] Sample: 52 leek germplasm accessions (Table 3, sourced from 5 provinces including Hebei and Gansu);

[0097] Testing process:

[0098] DNA was extracted (same as in Example 1);

[0099] The optimized system from Example 2 was used for amplification with SSR423 primers;

[0100] 8% polyacrylamide gel electrophoresis (160V), silver staining and development.

[0101] result:

[0102] Dormant germplasm (21-1, 21-3~21-5, etc.): No band at 177bp;

[0103] Non-dormant germplasm (21-2, 21-6, etc.): band at 177bp;

[0104] The identification results showed 100% consistency with the field phenotype.

[0105] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A chive SSR-PCR reaction system, comprising a reaction solution. Its characteristics are: The 20 μL reaction solution contains the following components: Template DNA 30 ng, Taq DNA polymerase 0.4 U, SSR primer pair 0.2 μM (per primer), Mg 2+ 2.0 mM, 0.2 mM dNTPs, 1× PCR buffer, with the remainder being ddH2O.

2. The SSR-PCR reaction technology system for chives according to claim 1, characterized in that: The PCR reaction procedure is as follows: 1) Pre-denaturation at 94℃ for 4 minutes; 2) Denaturation at 94℃ for 30 seconds, annealing at 57℃ for 30 seconds, extension at 72℃ for 45 seconds, for a total of 35 cycles; 3) 72℃ for a final extension of 7 minutes; 4) Store at 10℃.

3. An application of a chive SSR-PCR reaction technology system, characterized in that: In the detection of dormancy traits in chives, SSR molecular marker primers were used to perform PCR amplification on chive germplasm. The primer pairs were selected from any of the following groups: SSR005: Upstream primer TTTTCTTTTTggAgAAAgTgTC (SEQ ID NO:1), downstream primer CTTgTATCAgCCTTCTTCTTTg (SEQ ID NO:2); SSR170: Upstream primer CgATTCAAAAACTCAAATAAgC (SEQ ID NO:3), downstream primer TCTTgTAAgAAACgATCCAATC (SEQ ID NO:4); SSR249: Upstream primer TTTgCCTAAAgCTTgTAATgTT (SEQ ID NO:5), downstream primer ACAACACCACATTACACTgAgA (SEQ ID NO:6); SSR325: Upstream primer ACCAACAAATCAACTCAAAgAg (SEQ ID NO:7), downstream primer gTCCAAgCTCATCTgTTACATT (SEQ ID NO:8); SSR423: Upstream primer TTgAAgCATCCCATAATTAAAC (SEQ ID NO:9), downstream primer CAAATCATCACTACTAACgggT (SEQ ID NO:10); SSR478: Upstream primer TAAAAATCgCATCTTTATCCTg (SEQ ID NO:11), downstream primer AAggTTgTgAggTgAgTgTTAg (SEQ ID NO:12); SSR610: Upstream primer gACACTTgAgAgTggAAACAgA (SEQ ID NO:13), downstream primer CgTAggCTATgACgAAgAATAg (SEQ ID NO:14).

4. The application of the leek SSR-PCR reaction technology system according to claim 3, characterized in that: The SSR molecular marker is SSR423, and its amplified fragment size is 177 bp, wherein: No 177bp band was found in dormant germplasm; A 177bp band was present in the non-dormant germplasm.

5. The application of the leek SSR-PCR reaction technology system according to claim 4, characterized in that, Includes the following steps: (1) Extract DNA from the leek germplasm to be tested; (2) Using the reaction system and procedure described in claims 1-2, PCR amplification was performed with SSR423 primers; (3) Detection of amplification products by electrophoresis: If there is no band at 177bp, it is determined to be dormant germplasm; if there is a band at 177bp, it is determined to be non-dormant germplasm.

6. A method for identifying dormancy traits in leek germplasm resources, characterized in that, This includes using the SSR-PCR reaction technology system described in claims 1-2, performing PCR amplification of the leek germplasm DNA to be tested using SSR423 primers, and distinguishing between dormant and non-dormant phenotypes based on the presence or absence of a 177bp band.

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