DNA (deoxyribonucleic acid) rapid extraction method suitable for brassicaceous vegetable SSR (simple sequence repeat) and SNP (single nucleotide polymorphism) marker detection and application thereof

Abstract

The invention discloses a DNA (deoxyribonucleic acid) rapid extraction method suitable for SSR (simple sequence repeat) and SNP (single nucleotide polymorphism) marker detection of brassicaceous vegetables and application of the DNA rapid extraction method, and belongs to the field of biological breeding. The rapid DNA extraction method provided by the invention comprises the following steps: placing tissues of low-protein parts of brassicaceous vegetables in a porous DNA extraction plate, adding a DNA extraction solution, sealing, carrying out constant high-temperature treatment, centrifuging, and uniformly mixing, so as to obtain the extracted DNA. According to the DNA rapid extraction method suitable for brassicaceous vegetable SSR and SNP marker detection, the to-be-detected sample is directly placed in the porous DNA extraction plate, the DNA extraction process is completed in the same plate, and a tedious tube transferring or liquid transferring step is not needed. According to the whole extraction method, DNA extraction can be completed only through sampling, one-time liquid adding, one-time heating and one-time centrifuging, the operation process only needs 10-15 min, and DNA can completely meet the follow-up PCR amplification requirements of SSR markers and SNP markers.

Description

Technical Field

[0001] The present invention relates to the field of biological breeding, and in particular to a rapid DNA extraction method suitable for detecting SSR and SNP markers of cruciferous vegetables and an application thereof. Background Art

[0002] With the development of molecular biology, molecular marker technology provides a precise and rapid tool for breeding by detecting genetic variation at the DNA level, and plays an irreplaceable role in molecular-assisted breeding, such as genetic diversity analysis, germplasm resource evaluation, and kinship studies. Among them, SSR and SNP markers are two extremely important molecular marker technologies. SSR (Simple Sequence Repeat) markers, as a polymorphic marker based on microsatellite DNA sequences, have the characteristics of abundant quantity, wide distribution, multiple allelic variations, and co-dominance. They can provide rich genetic information and are suitable for population genetic structure analysis and genetic diversity research. SNP (Single Nucleotide Polymorphism) markers, as the smallest genetic variation unit in the genome, have the advantages of high density, strong representativeness, and good genetic stability. In addition, the detection methods are flexible and diverse, and can be easily automated. They are particularly suitable for large-scale genome association studies and molecular marker-assisted selection breeding.

[0003] In molecular breeding research, the key technical foundation is to quickly and efficiently complete DNA extraction and genotyping within a specific time. However, traditional DNA extraction methods involve the use of toxic reagents, are complex to operate, and are time-consuming. Impurities are also likely to be introduced during the tedious extraction process, resulting in the purity and quality of the extracted DNA failing to meet the requirements of marker detection. In addition, traditional DNA extraction methods often require professional reagents, laboratory equipment, and technicians, which not only increases costs but also limits its application in resource-limited areas or small-scale studies. Although the use of DNA extraction kits is quick to operate and extracts high-quality DNA, it is expensive. Especially in high-throughput detection, traditional DNA extraction methods and kit methods are difficult to meet the requirements of large-scale, safe, fast, and low-cost, which greatly limits the widespread application of molecular marker technology in biological breeding.

[0004] In order to overcome these limitations, the field of biotechnology has been seeking to improve DNA extraction methods to simplify the operating steps, improve extraction efficiency and reduce costs. The ideal DNA extraction method should be fast, efficient, low-cost, and able to provide DNA with the purity and quality required to meet the marker detection requirements. Such a method will greatly promote the application of molecular marker technology in biological breeding, improve breeding efficiency, accelerate the cultivation of new varieties, and also provide more accurate and reliable data support for genetic research. Therefore, the development of a rapid DNA extraction method suitable for SSR and SNP marker detection in cruciferous vegetables can not only solve the bottleneck problem in the existing technology, but also promote the development of biological breeding technology, which has important scientific and practical application value. Summary of the Invention

[0005] The purpose of the present invention is to provide a rapid DNA extraction method suitable for SSR and SNP marker detection in cruciferous vegetables and its application, so as to solve the problems of the existing technology of DNA extraction from cruciferous vegetables such as multiple steps, long time consumption, instability, poor extraction quality, and high cost. The extraction method has safe and simple operation steps, high extraction efficiency and is cost-effective. It is suitable for rapid DNA extraction for molecular marker detection of cruciferous vegetables such as cabbage, Chinese cabbage, and radish.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables, comprising the following steps:

[0008] Tissues from low-protein parts of cruciferous vegetables were placed in a porous DNA extraction plate;

[0009] Add DNA extraction solution to the DNA extraction plate, seal it and then perform constant high temperature treatment;

[0010] After the treatment is completed, the DNA extraction plate is taken out, and the mixture is centrifuged to obtain the DNA suitable for detecting SSR and SNP markers of cruciferous vegetables.

[0011] Furthermore, in the rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables, the DNA extraction plate is a 96-well plate or a 384-well plate with a volume of 0.1 mL to 2 mL.

[0012] Furthermore, in the rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables, the low-protein part of the cruciferous vegetables is the young leaf tissue of the plant to be tested or the part from the stem to the root hair after the seeds have been germinated for 2-3 days.

[0013] Furthermore, in the rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables, the DNA extraction solution is a 0.1 mol / L NaOH solution.

[0014] Furthermore, in the rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables, the amount of DNA extraction solution used in any extraction well in the DNA extraction plate is 100 μL.

[0015] Furthermore, in the rapid DNA extraction method suitable for detecting SSR and SNP markers of cruciferous vegetables, the sealing treatment condition is: using a 96-well or 384-well PCR plate matching silica gel cover to seal the DNA extraction plate.

[0016] Furthermore, in the rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables, the conditions for high-temperature treatment are: placing in a PCR instrument or a metal bath, setting the operating temperature to 94°C to 100°C, and the treatment time to 5min to 15min.

[0017] Preferably, the conditions for the high temperature treatment are: placing in a PCR instrument or a metal bath, setting the operating temperature to 94° C., and the treatment time to 10 minutes.

[0018] Furthermore, in the rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables, the centrifugal treatment conditions are: a rotation speed of 4800 rpm to 12000 rpm, and a centrifugation time of 30s to 300s.

[0019] Preferably, the centrifugal treatment conditions are: a rotation speed of 4800 rpm and a centrifugal time of 30 s.

[0020] The present invention provides an application of the rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables in molecular detection, molecular marker-assisted breeding or genetic map construction.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] The present invention provides a rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables. By placing the sample to be tested directly into a porous DNA extraction plate, the DNA extraction process is completed within the same plate, eliminating the need for cumbersome tube transfer or pipetting steps. The entire extraction method requires only sampling, one liquid addition, one heating step, and one centrifugation step to complete DNA extraction. The operation takes only 10-15 minutes, and the DNA fully meets the requirements for subsequent PCR amplification of SSR and SNP markers.

[0023] The rapid DNA extraction method of the present invention requires only three simple steps and eliminates the need for complex procedures, greatly simplifying the DNA extraction process. High extraction efficiency: By optimizing extraction conditions, the present invention can complete DNA extraction within 10-15 minutes, significantly improving extraction efficiency. Uniform purity: The DNA extracted by the present invention meets the purity requirements for SSR and SNP marker detection, eliminating the need for additional purification steps.

[0024] The rapid DNA extraction method provided by the present invention only requires the use of NaOH commonly found in laboratories, is low-cost, and does not require the purchase of expensive plant tissue genomic DNA extraction kits on the market.

[0025] The rapid DNA extraction method provided by this invention utilizes a multi-well DNA extraction plate. Due to the small capacity of each well, this helps reduce operational errors between samples and ensures consistent extracted DNA concentration. This eliminates the need for individual concentration adjustments for each sample, simplifying subsequent experimental procedures.

[0026] The rapid DNA extraction method provided by this invention uses a PCR plate as the DNA extraction plate. Compared to single centrifuge tube operations, it offers higher throughput, lower reagent consumption, and fixed sample well positions, reducing sampling confusion. Compared to common DNA extraction techniques such as the CTAB method and kit extraction, it offers the advantages of simplified procedures, high extraction efficiency, uniform purity, low toxicity, and low cost.

[0027] The rapid DNA extraction method provided by the present invention has good application effects in the fields of molecular detection of cruciferous materials such as cabbage, Chinese cabbage, and radish, molecular marker-assisted breeding, and genetic map construction. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the examples. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be considered as limiting the scope. A person of ordinary skill in the art can also derive other relevant drawings based on these drawings without inventive effort. In the drawings:

[0029] Figure 1 Photos of DNA extraction from different tissues of the radish variety Qixing provided in Example 1 of the present invention;

[0030] Figure 2 The SSR marker purity identification result of the radish variety Qixing provided in Example 1 of the present invention;

[0031] Figure 3The DNA extraction concentration test results of the cabbage variety Sulu 117 provided in Example 2 of the present invention;

[0032] Figure 4 The SSR marker purity identification result of the Chinese cabbage variety Sulu 117 provided in Example 2 of the present invention;

[0033] Figure 5 The DNA extraction concentration test results of the green stalk vegetable provided in Example 3 of the present invention; Figure 6 This is the molecular marker genotyping result of the core germplasm resource BrFLC1-SNP1 of the green onion provided in Example 3 of the present invention.

[0034] Figure 7 The results of DNA quality testing using spectrophotometer and agarose gel electrophoresis in the test examples, control example 1, and control example 2 of the present invention are shown. DETAILED DESCRIPTION

[0035] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. However, the specific experimental methods involved in the following examples, unless otherwise specified, are all conventional methods or are performed under the conditions recommended by the manufacturer's instructions.

[0036] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be purchased from the market.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0038] The present invention provides a rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables, comprising the following steps:

[0039] S1: Take the low-protein part of cruciferous vegetables and place them in a porous DNA extraction plate;

[0040] S2: Add DNA extraction solution to the DNA extraction plate, seal it and then perform constant high temperature treatment;

[0041] S3: After the treatment is completed, the DNA extraction plate is taken out, and the mixture is centrifuged to obtain the DNA suitable for the detection of SSR and SNP markers of cruciferous vegetables.

[0042] Furthermore, the DNA extraction plate is a 96-well plate or a 384-well plate of 0.1mL to 2mL. The specifications of the DNA extraction plate can be 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1mL, 1.2mL, 1.4mL, 1.6mL, 1.8mL, 2mL, etc., but are not limited to the listed values. Other values ​​not listed within this range are equally applicable. Preferably, the DNA extraction plate used in the present invention is a 96-well plate of 0.2mL, which is more suitable for most laboratory PCR instruments. The subsequent high-temperature treatment can be carried out by the PCR instrument with higher efficiency and faster speed than water bath heating. Because the sample tissue needs to be placed in a 0.2ml DNA extraction plate, it is smaller than the leaves or low-protein tissues used for extraction in traditional 2ml centrifuge tubes. In step S1, the sample tissue can be more accurately placed in the porous extraction plate by using tweezers. The tweezers method can ensure that the contact surface between the tool and the sample tissue is small enough, which can effectively avoid contamination of the sample tissue DNA.

[0043] Furthermore, the low-protein part of the cruciferous vegetable is the tender leaf tissue of the plant to be tested, or the area between the stem and the root hairs after the seeds have been germinated for 2-3 days. Because the DNA content in different plant tissues varies, young plant tissue generally contains very little protein, polysaccharides, and phenolic compounds. Furthermore, since cells are in a period of active division at this time, the DNA content is also relatively high. Therefore, almost all experimental tutorials recommend using young leaf tissue as the test material for DNA extraction. However, the growth time of the test material often reduces the overall efficiency of molecular testing. Therefore, we have conducted experiments using the same extraction method to test the quality of DNA obtained from different tissue parts such as plant leaves, radicles, plumules, and seeds. The results show that except for intact seeds, the concentration and purity of DNA extracted from leaves, radicles, and plumules can meet the requirements of ordinary PCR amplification. Analysis may be that the content of impurities such as protein in seeds is relatively high, which reduces the content of DNA extraction. On the other hand, the seed coat contains a variety of polyphenolic compounds such as phenolic acids, flavonoids and anthocyanins. They have strong oxidizing properties and can react with DNA, causing DNA degradation or precipitation, thereby reducing the extraction efficiency and DNA quality. Therefore, for seed DNA extraction, only the CTAB method and ready-made kits can obtain relatively pure DNA. The low-protein part of the cruciferous vegetable of the present invention is the young leaf tissue of the plant to be tested or the part between the stem and the root hair after the seeds are germinated for 2-3 days. The content of DNA in this part is low, and the content of other impurities is low, which can improve the quality of the extracted DNA.

[0044] Furthermore, the DNA extraction solution is a 0.1 mol / L NaOH solution. This DNA extraction solution is prepared by weighing 2 g of solid NaOH and adding ddH2O to a constant volume of 500 mL. The pH of the NaOH solution used in the present invention is 13. The high pH of the extraction solution ruptures the cell wall, denaturing DNA and proteins, allowing for direct extraction.

[0045] Furthermore, the amount of DNA extraction solution in any extraction well of the DNA extraction plate is 100 μL. The present invention uses a multi-channel pipette with a range of 100 μL to 1000 μL, preferably a 12-channel pipette with a range of 100 μL, which is more accurate and efficient.

[0046] Furthermore, the sealing treatment condition is: using a 96-well or 384-well PCR plate matching silica gel cover to seal the DNA extraction plate.

[0047] Furthermore, the conditions for the high temperature treatment are: placing in a PCR instrument or a metal bath, setting the operating temperature to 94°C to 100°C, and the treatment time to 5min to 15min. The operating temperature can be: 94°C, 94.5°C, 95°C, 95.5°C, 96°C, 96.5°C, 97°C, 97.5°C, 98°C, 98.5°C, 99°C, 99.5°C, 100°C, etc., but are not limited to the listed values. Other values ​​not listed in this range are also applicable. The treatment time can be 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, etc., but are not limited to the listed values. Other values ​​not listed in this range are also applicable. Preferably, the conditions for the high temperature treatment are: placing in a PCR instrument or a metal bath, setting the operating temperature to 94°C, and the treatment time to 10min.

[0048] Furthermore, the centrifugal treatment conditions are: a rotation speed of 4800 rpm to 12000 rpm, and a centrifugation time of 30 s to 300 s. The centrifugal speed can be 4800 rpm, 5000 rpm, 5500 rpm, 6000 rpm, 6500 rpm, 7000 rpm, 7500 rpm, 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm, 10000 rpm, 10500 rpm, 11000 rpm, 11500 rpm, 12000 rpm, etc., but is not limited to the values ​​listed above, and other values ​​not listed within this range are also applicable. The centrifugation time can be: 30s, 45s, 60s, 75s, 90s, 105s, 120s, 135s, 150s, 165s, 180s, 195s, 210s, 225s, 240s, 255s, 270s, 285s, 300s, etc., but is not limited to the values ​​listed above. Other values ​​not listed within this range are also applicable. Preferably, the centrifugation conditions are: a rotation speed of 4800 rpm and a centrifugation time of 30s.

[0049] The present invention provides a rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables. By placing the sample to be tested directly into a porous DNA extraction plate, the DNA extraction process is completed within the same plate, eliminating the need for cumbersome tube transfer or pipetting steps. The entire extraction method requires only sampling, one liquid addition, one heating step, and one centrifugation step to complete DNA extraction. The operation takes only 10-15 minutes, and the DNA fully meets the requirements for subsequent PCR amplification of SSR and SNP markers.

[0050] The rapid DNA extraction method provided by the present invention eliminates the need for transfer operations: the entire process from tissue processing to DNA extraction is completed directly in a 96 / 384-well plate, eliminating the tube transfer and pipetting steps of traditional methods (such as the CTAB method, which requires multiple transfers), reducing the risk of contamination and time consumption. The compatibility of equipment is optimized, and a standard PCR plate or metal bath is used for high-temperature treatment. It is compatible with conventional laboratory equipment and does not require a dedicated extraction instrument. The sampling tissue is optimized, and the stem-root hair transition zone after young leaves or seeds are germinated is used. This type of part has a low protein content and a thin cell wall, which significantly improves the lysis efficiency and avoids polysaccharide / polyphenol interference (compared to the traditional method, which requires a complex purification step). The chemical reagents and reaction system are simplified, and a low-concentration alkaline lysis solution is used for efficient replacement, requiring only a 0.1 mol / L NaOH solution, which not only eliminates the acid-base neutralization step but also achieves the effect of rapid release of DNA by high-temperature alkaline lysis, eliminating the use of toxic reagents, reducing costs and being safer. This reduces trace reagent consumption, requiring only 100 μL of lysis buffer per well. Combined with a multi-well plate design, 96 / 384 samples can be processed simultaneously, significantly saving reagent costs. This invention addresses the industry pain points of low efficiency, high cost, and cumbersome procedures in DNA extraction from cruciferous vegetables through a four-dimensional innovative extraction technology: "Clear sample selection - Reagent screening optimization - Operation simplification - Equipment adaptation." It is particularly suitable for large-scale marker detection scenarios in molecular breeding.

[0051] To further illustrate the present invention, the following describes a rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables and its application provided by the present invention in combination with examples. However, it should be understood that these examples are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided only to further illustrate the features and advantages of the present invention, rather than to limit the claims of the present invention. The scope of protection of the present invention is not limited to the following examples.

[0052] Example 1:

[0053] This example provides a rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables. DNA from different tissues of the sample was extracted and applied to an experiment to identify the purity of the radish variety Qixing using SSR markers. The specific steps are as follows:

[0054] 1. Sampling: Select radish (radish variety Qixing) material leaves, stems, embryos, and seeds, take a small amount of tissue sample with tweezers, and place it in a 0.2ml 96-well PCR plate (see Figure 1 ).

[0055] 2. Add DNA extraction solution: Add 100 μl of 0.1 mol / L NaOH solution to each well containing tissue sample.

[0056] 3. Heat treatment: Cover the 96-well plate with a plastic cover and place it in a PCR instrument. Set the temperature to 94°C and the time to 10 minutes.

[0057] 4. Centrifugation: After the heat treatment, remove the 96-well plate and centrifuge at 4800 rpm for subsequent PCR experiments.

[0058] 5. PCR amplification: PCR reaction system (10 μl): 1 μl DNA template, 9 μl premix, including 0.15 μl forward primer, 0.15 μl reverse primer, 5 μl 2× Taqmaster Mix, and 3.7 μl ultrapure water.

[0059] 6. PCR reaction procedure: ①95℃, 3 min; ②95℃, 30 s; ③57℃, 30 s; ④72℃, 30 s; ⑤Repeat ②③④.

[0060] 7. Repeat 35 cycles: ⑥ 72°C, 5 min; ⑦ 24°C, store. Make up to 20 μl with ultrapure water. PCR reaction procedure: pre-denaturation at 95°C.

[0061] 8. 3 min, 95 °C 30 s, 57 °C 30 s, 72 °C 30 s, 35 cycles, 72 °C 5 min.

[0062] DNA was extracted from different tissues of the radish variety Qixing in this embodiment, and the results are shown in FIG. Figure 1 The DNA extracted in this example was directly used to detect the purity of the radish variety Qixing using SSR markers: the bands were detected by non-denaturing polyacrylamide gel electrophoresis and silver staining technology, and the results were shown in FIG. Figure 2 shown.

[0063] from Figure 1 and Figure 2 The results show that DNA was extracted from different parts of radish, such as leaves, stems, embryos, and seeds, using the extraction method of the present invention. It was found that leaves, stems, and embryos could all be amplified normally, but some bands in the embryos were slightly dragged. Analysis showed that the embryo tissue contained secondary metabolites such as proteins and polysaccharides, which could not be completely removed, interfering with DNA migration and causing blurred bands. The seeds were completely bandless. Analysis of substances such as polyphenols contained in the seed coat inhibited the PCR reaction or interfered with electrophoretic migration. Therefore, the extraction method of the present invention is more suitable for tissue parts with low plant protein content and few polysaccharides and polyphenols. Using the above parts as test materials can significantly improve the lysis efficiency and avoid interference.

[0064] Example 2:

[0065] This example is a rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables. The method is used to extract DNA from Chinese cabbage and apply it to an experiment to identify the purity of the Chinese cabbage variety Sulu 117 using SSR markers. The specific steps are as follows:

[0066] 1. Sampling: Select cabbage (cabbage variety Speed ​​Green 117) material leaves, tear off a small amount of tissue sample with tweezers, and place it into a 0.2 ml 96-well PCR plate.

[0067] 2. Add DNA extraction solution: Add 100 μl of 0.1 mol / L NaOH solution to each well containing tissue sample.

[0068] 3. Heat treatment: Cover the 96-well plate with a plastic cover and place it in a PCR instrument. Set the temperature to 94°C and the time to 10 minutes.

[0069] 4. Centrifugation: After the heat treatment, remove the 96-well plate and centrifuge at 4800 rpm for subsequent PCR experiments.

[0070] 5. PCR amplification: PCR reaction system (10 μl): 1 μl DNA template, 9 μl premix, including 0.15 μl forward primer, 0.15 μl reverse primer, 5 μl 2× Taqmaster Mix, and 3.7 μl ultrapure water.

[0071] 6. PCR reaction procedure: ①95℃, 3 min; ②95℃, 30 s; ③57℃, 30 s; ④72℃, 30 s; ⑤Repeat ②③④.

[0072] 7. Repeat 35 cycles: ⑥ 72°C, 5 min; ⑦ 24°C, store. Make up to 20 μl with ultrapure water. PCR reaction procedure: pre-denaturation at 95°C.

[0073] 8. 3 min, 95 °C 30 s, 57 °C 30 s, 72 °C 30 s, 35 cycles, 72 °C 5 min.

[0074] In this example, the extracted DNA can be directly used to detect the purity of the cabbage variety Sulu 117 using SSR markers. The DNA quality and concentration were evaluated by ultra-micro spectrophotometer Bio Drop-duo. The results are shown in Figure 2. Figure 3 The obtained genomic DNA was amplified by PCR using SSR molecular markers, and the bands were detected by non-denaturing polyacrylamide gel electrophoresis and silver staining technology. The results are shown in Figure 4 shown.

[0075] from Figure 3 The results show that the concentration of DNA extracted in this embodiment reaches 81.05 μg / mL. Figure 4The results show that the SSR molecular markers can be used to effectively amplify the genomic DNA obtained by PCR, indicating that the DNA extracted by the present invention is of qualified quality and can meet the needs of conventional PCR reactions.

[0076] Example 3:

[0077] This example is a rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables. DNA from 52 core germplasm resources of Brassica rapa was extracted and SNP genotyping was performed on the materials using SNP markers. The specific steps are as follows:

[0078] 1. Sampling: Select 52 test material leaves (green cabbage), with one replicate for each test material. Use tweezers to tear off a small amount of tissue sample and place it into a 0.2 ml 96-well PCR plate.

[0079] 2. Add DNA extraction solution: Add 100ul of 0.1mol / LNaOH solution to each well containing tissue sample.

[0080] 3. Heat treatment: Cover the 96-well plate with a plastic cover and place it in a PCR instrument. Set the temperature to 94°C and the time to 10 minutes.

[0081] 4. Centrifugation: After the heat treatment, remove the 96-well plate and centrifuge at 4800 rpm for subsequent PCR experiments.

[0082] 5. PCR amplification: PCR reaction system (10 μl): 1 μl DNA template, 9 μl premix, including 0.15 μl forward primer, 0.15 μl reverse primer, 5 μl 2× Taqmaster Mix, and 3.7 μl ultrapure water.

[0083] 6. PCR reaction procedure: ①95℃, 3 min; ②95℃, 30 s; ③57℃, 30 s; ④72℃, 30 s; ⑤Repeat ②③④.

[0084] 7. Repeat 35 cycles: ⑥ 72°C, 5 min; ⑦ 24°C, store. Make up to 20 μl with ultrapure water. PCR reaction procedure: pre-denaturation at 95°C.

[0085] 8. 3 min, 95 °C 30 s, 57 °C 30 s, 72 °C 30 s, 35 cycles, 72 °C 5 min.

[0086] The DNA extracted in this example was amplified with the BrFLC1-SNP1 molecular marker primer and then directly used in the test of detecting the bolting resistance trait of the core germplasm resources of green stalk cabbage using SNP markers. The DNA quality and concentration were evaluated and tested by the ultra-micro spectrophotometer Bio Drop-duo. The results are shown in Figure 2. Figure 5The DNA was amplified by PCR after uniform concentration, and genotyping was performed by fluorescence quantitative instrument 7500. The results are shown in Figure 6 shown.

[0087] from Figure 5 It can be seen that the concentration of DNA extracted in this example reached 169.1 μg / mL. Figure 6 The results show that the rapid extraction method of the present invention can clearly genotype SNP markers after SNP marker amplification using a fluorescence quantitative instrument, and the consistency rate with the phenotype is over 98%, indicating that it can be applied to the identification of bolting-resistant SNP markers in the core germplasm resources of Brassica rapa.

[0088] Test example:

[0089] This test example provides a rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables. The specific steps are as follows:

[0090] 1. Use tweezers to take a small amount of tissue from the low-protein part of cruciferous vegetables and place it in a 0.2ml 96-well plate. DNA extraction can be performed directly in the plate, eliminating the need for tube transfer and pipetting.

[0091] 2. Use a 100ul 12-channel pipette to add 100ul of DNA extraction solution (0.1mol / LNaOH), seal the tube, and place it in a PCR instrument or metal bath for a constant high temperature treatment at 94°C for 10 minutes.

[0092] 3. After centrifugation at 4800 rpm for 30 seconds, remove the DNA extraction plate and centrifuge to mix. DNA extraction is now complete.

[0093] Comparative Example 1:

[0094] In the prior art, DNA extraction techniques for cruciferous vegetables mostly use the CTAB method and kit extraction method.

[0095] Control Example 1 is a kit extraction method in the prior art, using a magnetic bead DNA extraction kit. With a single person extracting 96 samples as the requirement, the time consumed by the extraction method of this test example and the extraction kit method of this control example 1 was statistically analyzed. The specific steps of the kit extraction method are as follows:

[0096] 1. Before use, add the required volume of anhydrous ethanol to the deproteinizing solution RD and the rinsing solution PWB according to the label on the bottle.

[0097] 2. Take about 100 mg of fresh plant tissue or about 30 mg of dry weight tissue, add liquid nitrogen and grind thoroughly.

[0098] 3. Quickly transfer the ground powder to a centrifuge tube pre-filled with 400 μl of GPM buffer and 5 μl of RNase A (10 mg / ml). Quickly invert to mix thoroughly. Place the tube in a 70°C water bath for 10 minutes, inverting the tube several times to mix the sample. For extracting plant tissues rich in polysaccharides and polyphenols, use GPM buffer plus.

[0099] 4. Centrifuge at 12,000 rpm for 4 minutes and transfer 300 μl of supernatant to a new centrifuge tube.

[0100] 5. Add 300 μl of buffer GHB, 300 μl of isopropanol and 15 μl of magnetic bead suspension G, shake to mix, and let it stand at room temperature for 5 minutes.

[0101] 6. Place the centrifuge tube on the magnetic stand and let it stand for 1 minute. When the magnetic beads are completely adsorbed, carefully remove the liquid.

[0102] 7. Remove the centrifuge tube from the magnetic stand, add 500 μl of deproteinization solution RD and shake to mix for 30 seconds.

[0103] 8. Place the centrifuge tube on the magnetic stand and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, carefully remove the liquid.

[0104] 9. Add 600 μl of PWB rinse buffer and shake to mix for 30 seconds.

[0105] 10. Place the centrifuge tube on the magnetic stand and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, carefully remove the liquid.

[0106] 11. Repeat steps 9 and 10 once.

[0107] 12. Place the centrifuge tube on a magnetic rack and dry it at room temperature for 10-15 minutes.

[0108] 13. Remove the centrifuge tube from the magnetic stand, add 50-100 μl of elution buffer TB, shake to mix, and incubate at 65°C for 3 minutes.

[0109] 14. Place the centrifuge tube on the magnetic rack and let it stand for 1 minute. After the magnetic beads are completely adsorbed, carefully transfer the DNA solution to a new centrifuge tube and store it under appropriate conditions.

[0110] Taking 96 samples as an example, the experimental operation time of the prior art extraction kit method in this control example 1 is 1 hour, while the experimental operation time of the extraction method in the test example of the present invention is 15 minutes, which is only 1 / 4 of the time taken by the kit method, greatly shortening the extraction time and improving the extraction efficiency.

[0111] Comparative Example 2:

[0112] Comparative Example 2 is the CTAB method in the prior art. Taking the extraction of 96 samples by a single person as the requirement, the experimental operation time of the extraction method of Experimental Example 1 and the CTAB method of Comparative Example 2 was statistically analyzed. The specific operation steps of the CTAB method are as follows:

[0113] 1. Take about 0.2g of plant true leaves from the field and place them in a 96-deep-well plate. Add 2 steel balls and 400μL of 2% CTAB into each well.

[0114] 2. Extract the buffer solution. Grind thoroughly with a high-speed grinder at 65 Hz for 75 seconds, place in a 65°C water bath for 30 minutes, and shake gently every 10 minutes.

[0115] 3. Add 400 μL of chloroform-isoamyl alcohol extract (24:1) to the deep-well plate and shake gently for 5 minutes.

[0116] 4. Centrifuge at 12000rpm for 5 minutes.

[0117] 5. Pipette 200 μl of the supernatant into a new 96-well PCR plate, add 200 μl of pre-cooled isopropanol, mix well, and place in a -20°C refrigerator for 20 min.

[0118] 6. Centrifuge at 12000 rpm for 10 min.

[0119] 7. Pour off the supernatant, add 150 μL of pre-cooled ethanol, cover with the rubber cover and turn the 96-well PCR plate upside down.

[0120] 8. Centrifuge at 12000rpm for 5 minutes.

[0121] 9. Discard the supernatant decisively and let the DNA dry at room temperature for 2 hours.

[0122] 10. Add 100 μL of ddH2O to dissolve the DNA and set aside.

[0123] Taking 96 samples as an example, the experimental operation of the prior art CTAB method in this comparative example 2 takes 90 minutes, while the experimental operation of the extraction method in the test example of the present invention takes 15 minutes, which is only 1 / 6 of the time taken by the CTAB method.

[0124] Result analysis:

[0125] The DNA quality and concentration of the test example, control example 1 and control example 2 were evaluated by ultra-micro spectrophotometer Bio Drop-duo. The results are shown in the table. Figure 2 .

[0126] The DNA samples obtained from the test example, control example 1, and control example 2 were subjected to 1% agarose gel electrophoresis (electrophoresis buffer: 1×TAE), stained with GelRed, and imaged and observed on a gel imager. The specific steps for agarose gel electrophoresis are as follows:

[0127] 1. Preparation of gel: Weigh 0.2 g agarose and dissolve it in 20 mL 1×TAE solution. Heat until the solution is clear. When the temperature drops to 60°C, add 1.5 μL of dye, shake gently to mix, insert a comb, and pour the gel.

[0128] 2. Sample application: Let it stand for 10-15 minutes. After the gel solidifies, remove the comb and use a pipette to mix 3 μL of DNA and buffer for sample application.

[0129] 3. Electrophoresis: After the sample is spotted, turn on the power supply and set the voltage to 120V. Turn off the electrophoresis switch when the indicator runs to the middle of the gel.

[0130] 4. Imaging: After electrophoresis, remove the gel and observe it in a UV gel imager.

[0131] The results of agarose gel electrophoresis on the DNA quality of the test example, control example 1 and control example 2 are shown in the figure. Figure 7 shown.

[0132] like Figure 7 As shown, Figure 7 -A (see Figure 7 (Upper left) is the test result of DNA extraction using Tiangen 50 times magnetic bead method plant genomic DNA extraction kit DP342 in control example 1: DNA concentration was 2055.8ug / ul, A260 / A280 was 1.810, between 1.8-2.0, and A260 / A230 was 2.066, between 2.0-2.5. Agarose gel electrophoresis bands (see Figure 7 Lower right A) is clear, bright, and uniform, indicating low impurities in the DNA, high purity, and good concentration consistency. However, the price is 760 yuan / 50 samples, which is not suitable for molecular marker screening and detection tests with large sample sizes.

[0133] like Figure 7 As shown, Figure 7 -B (see Figure 7 (Upper right) is the test result of DNA extracted from cabbage leaves using the extraction method of the present invention: the DNA concentration was 3309.3ug / ul, A260 / A280 was 1.288, between 1.0-1.5, and A260 / A230 was 1.389, between 1.0-1.5. Figure 7 Lower right B) Slightly bright and uniform, indicating that there are many impurities such as phenols in the DNA and the concentration is low.

[0134] like Figure 7 As shown, Figure 7 -C (see the lower left of the figure) is the result of DNA test of cabbage leaves extracted by CTAB method in 2ml centrifuge tube in control example 2: DNA concentration was 3043.9ug / ul, A260 / A280 was 1.719, close to 1.8-2.0, and A260 / A230 was 1.909, close to 2.0-2.5. Agarose gel electrophoresis bands (see Figure 7 Lower right (C) shows both light and dark areas, indicating relatively high DNA purity, but is not suitable for experiments requiring strict uniformity in DNA concentration.

[0135] In summary, the present invention provides a rapid DNA extraction method suitable for the detection of SSR and SNP markers in cruciferous vegetables, which has the advantages of good uniformity of extracted DNA concentration, simple operation, low toxicity, and low cost.

[0136] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables, characterized in that: The following steps are involved: Tissues from low-protein parts of cruciferous vegetables were placed in a porous DNA extraction plate; Add DNA extraction solution to the DNA extraction plate, seal it and then perform constant high temperature treatment; After the treatment is completed, the DNA extraction plate is taken out, and the mixture is centrifuged to obtain the DNA suitable for detecting SSR and SNP markers of cruciferous vegetables.

2. The rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables according to claim 1, characterized in that: The DNA extraction plate is a 96-well plate or a 384-well plate with a capacity of 0.1 mL to 2 mL.

3. The rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables according to claim 1, characterized in that: The low-protein part of the cruciferous vegetable is the young leaf tissue of the plant to be tested or the part between the stem and the root hair after the seeds are germinated for 2-3 days.

4. The rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables according to claim 1, characterized in that: The DNA extraction solution is 0.1 mol / L NaOH solution.

5. The rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables according to claim 4, characterized in that: The amount of DNA extraction solution in any extraction well of the DNA extraction plate is 100 μL.

6. The rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables according to claim 1, characterized in that: The sealing treatment conditions are: using a 96-well or 384-well PCR plate-matched silica gel cover to seal the DNA extraction plate.

7. The rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables according to claim 1, characterized in that: The conditions for the high temperature treatment are: placing in a PCR instrument or a metal bath, setting the operating temperature to 94° C. to 100° C., and the treatment time to 5 min to 15 min.

8. The rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables according to claim 7, characterized in that: The conditions for the high temperature treatment are: placing the tube in a PCR instrument or a metal bath, setting the operating temperature to 94° C., and treating for 10 minutes.

9. The rapid DNA extraction method suitable for detecting SSR and SNP markers in cruciferous vegetables according to claim 1, characterized in that: The conditions of the centrifugal treatment are: a rotation speed of 4800 rpm to 12000 rpm, and a centrifugal time of 30s to 300s.

10. Use of the rapid DNA extraction method suitable for SSR and SNP marker detection in cruciferous vegetables according to any one of claims 1 to 9 in molecular detection, molecular marker-assisted breeding or genetic map construction.

Images