Extracting solution and kit for extracting DNA (Deoxyribonucleic Acid) of sorghum seeds as well as extracting method and application of extracting solution

Abstract

The invention belongs to the technical field of biology, and particularly relates to an extracting solution for extracting DNA of sorghum seeds, a kit and an extracting method and application of the extracting solution. In order to solve the technical problems that the existing sorghum DNA extraction method is complicated, the extraction efficiency is low and long-term preservation cannot be realized, the invention provides a sorghum seed DNA extracting solution which comprises a DNA quick extracting solution and a cleaning solution, the rapid extracting solution contains Tris-HCl, NaCl, Tween-20 and EDTA (Ethylene Diamine Tetraacetic Acid); and the cleaning solution contains Tris-HCl and Tween-20 (polysorbate-20). By combining the extracting solution or the prepared kit with cellulose filter paper treated by NaCl, the DNA of the sorghum seeds can be rapidly extracted within 20 seconds, the extraction quality is high, the DNA of the sorghum seeds can be preserved for a short time, and meanwhile, the method has no inhibition effect on downstream PCR (Polymerase Chain Reaction). Therefore, the extracting solution, the kit and the extracting method can be quickly applied to fields or detection sites.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an extraction solution, kit, extraction method, and application for extracting DNA from sorghum seeds. Background Technology

[0002] Sorghum is an annual herbaceous plant belonging to the genus Sorghum in the family Poaceae, and it is the world's fifth largest cereal crop. As a highly resilient C4 crop, sorghum can adapt to harsh environments such as drought, salinity, and poor soil. Its grains are rich in starch, protein, dietary fiber, and various functional components, making it not only an important food source for humans but also a high-quality raw material for animal feed. Furthermore, it plays an irreplaceable role in the brewing and energy sectors, including liquor, beer, and fuel ethanol production.

[0003] With the rapid development of molecular breeding technology, the genetic improvement and variety innovation of sorghum have placed higher demands on gene-level research. DNA extraction from sorghum seeds is a necessary foundation for conducting work such as variety purity identification, molecular marker development, gene mapping, and genetic map construction. However, compared to leaves and roots, sorghum seeds contain a large amount of starch, protein, lipids, and phenolic compounds. These substances can easily interfere with the purity of the final product during DNA extraction, seriously affecting subsequent molecular biology operations such as PCR amplification and gene sequencing.

[0004] Current plant DNA extraction methods (such as the CTAB method) have significant limitations when processing sorghum seeds: the traditional CTAB method requires multiple chloroform-isoamyl alcohol extractions, which is cumbersome and time-consuming, and the use of toxic reagents poses potential health risks to operators; while commercial DNA extraction kits can simplify the process to some extent, they are expensive, and most products are not effective at removing the high starch and protein content in cereal seeds, affecting the results of subsequent experiments. Furthermore, existing methods largely rely on laboratory equipment such as centrifuges and constant-temperature water baths, making them difficult to apply rapidly in the field or at testing sites. Therefore, developing a highly efficient DNA extraction solution formulation and a corresponding rapid extraction method specifically tailored to the characteristics of sorghum seeds is of great significance for promoting the popularization of sorghum molecular breeding technology and improving the efficiency of variety identification. Summary of the Invention

[0005] To address the technical challenges of existing sorghum DNA extraction methods being complex, inefficient, and unable to be preserved long-term, this invention provides a combined extraction solution and kit that offers mild lysis conditions, simple operation, high DNA extraction quality, and short-term preservation of sorghum seed DNA, as well as an extraction method and application for sorghum seed DNA.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, the present invention provides an extraction solution for extracting DNA from sorghum seeds, comprising a DNA rapid extraction solution and a DNA washing solution; wherein the rapid extraction solution contains tris-HCl, sodium chloride (NaCl), Tween-20 and ethylenediaminetetraacetic acid (EDTA); and the washing solution contains Tris-HCl and Tween-20.

[0007] Furthermore, the rapid extraction solution contains Tris-HCl 50–65 mmol / L, NaCl 50–150 mmol / L, Tween-20 0.5–2.0% v / v and EDTA 10 mmol / L.

[0008] Furthermore, the pH of the rapid extraction solution is 7.5–8.5.

[0009] Furthermore, the cleaning solution contains 10–20 mmol / L Tris-HCl and 0.1–0.5% v / v Tween-20.

[0010] Furthermore, the pH of the cleaning solution is 7.5–8.5.

[0011] Furthermore, the pH of the rapid extraction solution and the washing solution is adjusted using sodium hydroxide (NaOH) solution.

[0012] Secondly, the present invention provides a kit for extracting DNA from sorghum seeds, which contains the above-mentioned extract for extracting DNA from sorghum seeds.

[0013] Thirdly, the present invention provides a method for extracting DNA from sorghum seeds, comprising the following steps: The filter paper, after being treated with NaCl aqueous solution, is dipped into the rapid extraction solution containing sorghum seeds, and then dipped into the washing solution to obtain filter paper adsorbed with sorghum seed DNA, thus completing the extraction of sorghum seed DNA.

[0014] Furthermore, the filter paper treated with NaCl solution is obtained by soaking medium-speed qualitative cellulose filter paper in a NaCl aqueous solution with a concentration of 20-60 g / L for ≥60 s and then drying it.

[0015] Furthermore, the sorghum seed comprises sorghum seed coat, endosperm, and / or whole seed.

[0016] Furthermore, sorghum seed powder is added to the quick extract at a ratio of 50:1 to 5:1 g / L.

[0017] Furthermore, the extraction time is ≤20 s.

[0018] Furthermore, the extraction method also includes using the extracted DNA as a DNA template to perform a PCR molecular reaction to amplify the DNA.

[0019] Preferably, the PCR reaction system also includes PCR Master Mix, primers for the conserved sorghum sequence, and water.

[0020] More preferably, the nucleotide sequences of the primers preceding and following the conserved sorghum sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0021] Preferably, the PCR reaction conditions are: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 51.5℃ annealing for 30 s, 72℃ extension for 20 s, for 35 cycles; 72℃ complete extension for 10 min.

[0022] Fourthly, the present invention provides the application of the above-mentioned extraction solution, kit or extraction method in plant DNA extraction.

[0023] Furthermore, the plants mentioned include, but are not limited to, sorghum, corn, barley, glutinous rice, and wheat.

[0024] Beneficial effects: This invention provides an extract of sorghum seed DNA, reagents containing the extract, and an extraction method, which have the following advantages compared with the prior art: 1. Simple operation and mild lysis conditions: The extraction solution of this invention can fully lyse sorghum seed cells within 20 seconds at 0-40℃ to extract DNA, and the whole process is completed in a centrifuge tube without the need for additional high-speed centrifugation, filtration and settling.

[0025] 2. The extraction solution of this invention has no inhibitory effect on downstream PCR reactions: DNA extracted using the combined extraction solution of this invention and cellulose filter paper pretreated with NaCl can be directly used for downstream PCR reactions, and the target band is amplified normally, without any inhibitory effect on the PCR reaction.

[0026] 3. The DNA extracted by the rapid extraction solution of the present invention can be preserved for a short period of time: DNA extracted using the combined extraction solution of the present invention and pretreated cellulose filter paper can be stored in the original DNA rapid extraction solution at room temperature for more than 72 hours and can still be directly used for downstream PCR reactions. The amplified target band does not show diffusion or dragging phenomenon. Attached Figure Description

[0027] Figure 1The image shows an agarose gel electrophoresis diagram of the PCR amplification products obtained by extracting DNA from sorghum seed coat, endosperm, and whole seed powder using the method of the present invention in Example 1, with the DNA as a template. Lanes 1-1, 1-2, and 1-3 represent three replicates of the sorghum seed coat powder extract, lanes 2-1, 2-2, and 2-3 represent three replicates of the sorghum endosperm powder, and lanes 3-1, 3-2, and 3-3 represent three replicates of the whole seed powder. M represents a DNA marker of 100–600 bp.

[0028] Figure 2 This is an agarose gel electrophoresis image of PCR amplification products obtained from whole sorghum seed powder using different temperature combinations of extraction solutions in Example 2, with the DNA used as a template. Lanes 1 to 9 represent the PCR amplification products of DNA extracted from combinations of extraction solutions at 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, and 40℃, respectively, and M is a DNA marker of 100–600 bp.

[0029] Figure 3 The image shows an agarose gel electrophoresis diagram of the PCR amplification products of whole sorghum seed powder DNA extracted using the method of the present invention and stored in the rapid extraction solution for different times in Example 3; where lanes 0 h, 24 h, 48 h, and 72 h represent storage times of 0 h, 24 h, 48 h, and 72 h in the rapid extraction solution, respectively, and M is a DNA marker of 100–600 bp.

[0030] Figure 4 The image shows agarose gel electrophoresis of PCR amplification products of whole sorghum seed powder extracted using different methods in Comparative Example 1. Lane 1 represents the traditional CTAB method, lane 2 represents the method of this invention, lane 3 represents the rapid extraction kit method, and M represents a DNA marker of 100–600 bp.

[0031] Figure 5 The image shows the electrophoresis results of PCR products extracted from sorghum seeds after treating medium-speed qualitative filter paper with different concentrations of NaCl solution in Comparative Example 2. Lanes 1, 2, 3, and 4 correspond to NaCl solution treatments of 60 g / L, 40 g / L, 20 g / L, and 0 g / L, respectively, and M is a DNA marker ranging from 100 to 600 bp. Detailed Implementation

[0032] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0033] In one embodiment of the present invention, an extraction solution for extracting DNA from sorghum seeds is provided, comprising a DNA rapid extraction solution and a DNA washing solution; the rapid extraction solution contains Tris-HCl, NaCl, Tween-20 and EDTA; the washing solution contains Tris-HCl and Tween-20.

[0034] In the above extraction solution, Tween-20 is used to lyse cell membranes and release DNA; NaCl is used to precipitate DNA; EDTA inhibits enzyme activity to ensure that DNA is not degraded; and Tris-HCl is used to buffer the pH.

[0035] In one specific embodiment of the present invention, the pH of the rapid extraction solution is 7.5–8.5. The pH of the cleaning solution is 7.5–8.5.

[0036] In a preferred embodiment of the present invention, the pH of both the rapid extraction solution and the washing solution is 8.0. This is because Tris-HCl and EDTA can exert their maximum effect at this pH.

[0037] In one embodiment of the present invention, the extract of sorghum seed DNA described above can be used to prepare a kit for extracting sorghum seed DNA for mass production and practical application.

[0038] In one embodiment of the present invention, a method for extracting DNA from sorghum seeds is provided, comprising the following steps: The filter paper, after being treated with NaCl aqueous solution, is dipped into the rapid extraction solution containing sorghum seeds, and then dipped into the washing solution to obtain filter paper adsorbed with sorghum seed DNA, thus completing the extraction of sorghum seed DNA.

[0039] In the above extraction method, the purpose of treating the filter paper with NaCl aqueous solution is to keep the DNA in a high-salt environment when it is adsorbed by the cellulose filter paper in the extraction solution, so as to prevent the DNA from re-dissolving.

[0040] In one embodiment of the present invention, the application of the above-described extraction solution, kit, or extraction method in plant DNA extraction is also provided.

[0041] The extraction of sorghum seed DNA using the above-described extraction solution, kit, or method is merely one successful example of this invention, and not the only exclusive option. Based on the teachings of this invention, the above-described extraction solution, kit, or method is also applicable to other plants, including but not limited to sorghum, corn, barley, glutinous rice, and wheat.

[0042] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0043] Example 1: Rapid extraction of DNA from sorghum seeds using the method of the present invention 1. Preparation of rapid extraction solution, washing solution, and filter paper for DNA extraction from sorghum seeds. Preparation of sorghum seed DNA rapid extraction solution (per 1 L): Tris-HCl 7.88 g (50 mmol / L), sodium chloride 5.84 g (100 mmol / L), Tween-20 10 mL (1% v / v), EDTA 2.92 g (10 mmol / L). The solution was prepared using ultrapure water, and the pH of the rapid extraction solution was adjusted to 8.0 using 40 g / L sodium hydroxide (NaOH).

[0044] Preparation of DNA washing solution from sorghum seeds (per 1 L): 1.57 g Tris-HCl (10 mmol / L), 1 mL Tween-20 (0.1% v / v), prepared with ultrapure water, and the pH of the washing solution was adjusted to 8.0 with 40 g / L NaOH solution.

[0045] Pretreatment of medium-speed qualitative cellulose filter paper: Immerse the medium-speed qualitative cellulose filter paper completely in 40 g / L NaCl solution for 1 min, remove and dry, and cut into strips 3 mm wide and 40 mm long for later use.

[0046] 2. Extract DNA from sorghum seed coat, endosperm, and whole seeds. DNA was extracted from sorghum seed coat, endosperm, and whole seeds using the rapid extraction solution, washing solution, and filter paper obtained in step 1. The specific steps are as follows: (1) Pretreatment of the sample to be extracted: 100 g of whole 'Yinuohong No. 4' sorghum seeds were ground using a rice milling machine. Only the powder obtained in the first 2 seconds was collected as seed coat powder, and only the powder obtained in the 6th to 10th seconds was collected as endosperm powder. All the powder produced within 10 seconds was collected as whole seed powder.

[0047] (2) Extraction process: Take 50 mg of seed coat, endosperm, and whole seed powder and place them into 1.5 mL centrifuge tubes respectively. Add 1 mL of the rapid extraction solution prepared in step 1 to each tube and shake for 10 s. Then, use the filter paper pretreated in step 1 to dip into the rapid extraction solution containing the seed coat, endosperm, and whole seed powder respectively. Remove the filter paper after 3 s. Then, dip the filter paper with the rapid extraction solution into the washing solution and remove it after 3 s. DNA extraction can be completed within 20 s. The DNA is adsorbed onto the filter paper and can be directly dipped into the PCR system again to be added as a DNA template for the next molecular reaction.

[0048] (3) Compare the DNA extraction effects of sorghum seed coat, endosperm, and whole seed powder.

[0049] Following the method described in step (2), DNA was extracted from the seed coat, endosperm, and whole seed powder of 'Yinuohong No. 4' sorghum seeds at room temperature of 26°C using pretreated filter paper, and the DNA was used as a template for PCR molecular reaction.

[0050] The PCR molecular reaction system consisted of 50 μL of PCR Master Mix (10103ES08, Yisheng Biotechnology Shanghai Co., Ltd., China), 21 μL of ultrapure water, and 2 μL each of the pre- and post-primer sequences of the conserved sorghum sequence. SEQ ID NO: 1: 5'-AGATGCCAACTATTCAAGACGA-3'; SEQ ID NO: 2: 5'-CCGCTAACTAAGAGAATGCAA-3'.

[0051] The PCR program was set as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 51.5℃ annealing for 30 s, 72℃ extension for 20 s, for 35 cycles; 72℃ final extension for 10 min. 4 μL of the obtained PCR amplification product was used for 2% agarose gel electrophoresis analysis. See details below. Figure 1 Lanes 1-1, 1-2, and 1-3 are three replicates of sorghum seed coat powder extract, lanes 2-1, 2-2, and 2-3 are three replicates of sorghum endosperm powder, lanes 3-1, 3-2, and 3-3 are three replicates of whole seed powder, and M is a DNA marker of 100–600 bp.

[0052] Depend on Figure 1 The experimental results showed that all nine samples could amplify specific bands of 400-500 bp with normal brightness and morphology, indicating that the extraction method of the present invention can rapidly extract DNA from sorghum seed coat, endosperm or whole seed within 20 seconds, and the DNA quality is good enough for PCR reaction.

[0053] Example 2: DNA extraction effect of combined extraction solutions at different temperatures on whole sorghum seed powder Using a water bath and a refrigerator, the rapid extract and washing solution from Example 1 were subjected to a water gradient cooling bath or heated to 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C, respectively, for treatments 1-9. See details. Figure 2 In the figure, lanes 1 to 9 represent the PCR amplification products of DNA extracted from combined extraction solutions at 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, and 40℃, respectively. M is a DNA marker of 100–600 bp. Following the method for extracting DNA from whole seeds as described in Example 1, DNA extraction from whole seed powder of “Yinuohong No. 4” was completed using combined extraction solutions at different temperatures (including rapid extraction solution and washing solution at the same temperature). This DNA was then used as a template for PCR reaction and agarose gel electrophoresis as described in Example 1.

[0054] Depend on Figure 2 The experimental results showed that all nine treatments could amplify specific bands of 400–500 bp with normal brightness and morphology, indicating that the extraction method of the present invention can rapidly extract sorghum seed DNA at common operating temperatures of 0–40℃, and the DNA quality is good enough for PCR reactions.

[0055] Example 3: The preservation effect of the DNA rapid extraction solution of the present invention on the extracted sorghum seed DNA at room temperature (26°C) Following the extraction method described in step 2 of Example 1, DNA was extracted from whole seed powder of 'Yinuohong No. 4' at room temperature. The extracted DNA was stored in DNA rapid extraction buffer for 0 h, 24 h, 48 h, and 72 h. The DNA stored for 0 h, 24 h, and 48 h was temporarily frozen at -20°C. After 72 h, the DNA from different storage times was used as a template, and the PCR reaction and agarose gel electrophoresis were performed again according to the method described in Example 1.

[0056] Depend on Figure 3 The experimental results show that DNA templates preserved in the DNA rapid extraction solution for 0 h, 24 h, 48 h, and 72 h can all amplify bands with consistent brightness and shape, indicating that the DNA rapid extraction solution of the present invention can effectively preserve the DNA extracted by the present invention at room temperature for at least 72 h.

[0057] Comparative Example 1: Experiment comparing DNA extraction from sorghum seeds using different extraction methods DNA was extracted from whole sorghum seed powder of 'Yinuohong No. 4' using the DNA rapid extraction buffer and washing solution described in Example 1 in conjunction with the rapid extraction method described in Example 1. The extraction effect and time required were compared with those of conventional CTAB extraction method and rapid DNA extraction kit (KG230630, Tiangen Biotech Co., Ltd., Beijing).

[0058] The method of this invention: DNA was extracted from whole sorghum seeds according to the method described in Example 1, and then PCR reaction and agarose gel electrophoresis were performed using a PCR system. The results are shown in [Figure 1]. Figure 4 Lane 2 (middle)

[0059] CTAB extraction method: 50 mg of whole sorghum powder of 'Yinuohong No. 4' was placed in a 1.5 mL centrifuge tube, and 500 μL of CTAB lysis buffer was added. The mixture was incubated at 65℃ for 60 min, with gentle shaking several times during incubation. Then, 500 μL of a phenol-chloroform-isoamyl alcohol (25:24:1 (v / v)) mixture was added, and the mixture was shaken at room temperature. After centrifugation, the supernatant was transferred to a clean 1.5 mL centrifuge tube. This operation was repeated twice. Pre-chilled isopropanol solution was added, and the mixture was precipitated at -20℃ for 2 hours. After centrifugation, the DNA precipitate was washed twice with 70% ethanol, dried, and dissolved in 500 μL of TE buffer. 1 μL of the DNA solution was used as a DNA template, and PCR and agarose gel electrophoresis were performed according to the method described in Example 1. The results are shown in [Figure 1]. Figure 4 Lane 1, Middle

[0060] Rapid Extraction Kit: Following the kit instructions, place 50 mg of whole seed powder into a 1.5 mL centrifuge tube, add 100 μL of buffer B1 to completely cover the sample, then add 100 μL of buffer B2, vortex to mix, and centrifuge at 12000 rpm for 2 min. After centrifugation, carefully aspirate 1 μL of the supernatant as a DNA template, and perform PCR and agarose gel electrophoresis as described in Example 1. The results are shown in [Figure 1]. Figure 4 Lane 3 (middle)

[0061] The results showed that the traditional CTAB method could not extract DNA of usable quality for PCR from whole sorghum seed powder. However, the method of this invention can extract DNA of usable quality for PCR, with extraction results comparable to commercial kits, while significantly improving extraction efficiency from over 2 minutes to within 20 seconds.

[0062] Comparative Example 2: Effect of medium-speed qualitative cellulose filter paper treated with different NaCl concentrations on DNA extraction from whole sorghum seeds The only difference from Example 1 is that, in the pretreatment process of the medium-speed qualitative cellulose filter paper, only the concentration of NaCl solution was modified in a gradient manner to 0 g / L, 20 g / L, 40 g / L, and 60 g / L. All other operations were the same as in Example 1. The differences in the DNA extraction effect of the medium-speed qualitative cellulose filter paper treated with four different NaCl concentrations on the whole seed powder of 'Yinuohong 4' were compared. The experimental results of sorghum DNA extraction from medium-speed qualitative filter paper treated with 60 g / L, 40 g / L, 20 g / L, and 0 g / L NaCl solutions are shown in the figures below. Figure 5 Lanes 1, 2, 3, and 4.

[0063] The results showed that the extraction effect of filter paper strips that were not completely immersed in NaCl solution was significantly worse than the other three treatments. Meanwhile, when the NaCl solution concentration was 40 g / L, the brightness of the amplified product was slightly higher than that of 20 g / L or 60 g / L, indicating that 40 g / L NaCl solution is the optimal concentration for treating filter paper strips and is the necessary solvent and concentration for the medium-speed qualitative cellulose filter paper treatment of this invention.

[0064] It should be noted that the specific features, structures, materials, or characteristics described in this specification may be combined in any suitable manner in one or more embodiments. Furthermore, those skilled in the art can combine and integrate the different embodiments described in this specification and the features of those embodiments without contradiction.

Claims

1. An extract for extracting DNA from sorghum seeds, characterized in that, It includes a DNA rapid extraction buffer and a DNA washing buffer; the rapid extraction buffer contains Tris-HCl, NaCl, Tween-20 and EDTA; the washing buffer contains Tris-HCl and Tween-20.

2. The extract for extracting DNA from sorghum seeds according to claim 1, characterized in that, The rapid extraction solution contains Tris-HCl 50–65 mmol / L, NaCl 50–150 mmol / L, Tween-20 0.5–2.0% v / v and EDTA 10 mmol / L; furthermore, the pH of the rapid extraction solution is 7.5–8.

5.

3. The extract for extracting DNA from sorghum seeds according to claim 1, characterized in that, The cleaning solution contains 10–20 mmol / L Tris-HCl and 0.1–0.5% v / v Tween-20; furthermore, the pH of the cleaning solution is 7.5–8.

5.

4. A kit for extracting DNA from sorghum seeds, characterized in that, An extract containing the DNA extraction solution for sorghum seeds as described in any one of claims 1 to 3.

5. A method for extracting DNA from sorghum seeds, characterized in that, Includes the following steps: The filter paper, after being treated with NaCl aqueous solution, is dipped into the rapid extraction solution containing sorghum seeds, and then dipped into the washing solution to obtain filter paper adsorbed with sorghum seed DNA, thus completing the extraction of sorghum seed DNA.

6. The method for extracting DNA from sorghum seeds according to claim 5, characterized in that, The filter paper treated with NaCl solution is obtained by soaking medium-speed qualitative cellulose filter paper in a NaCl aqueous solution with a concentration of 20-60 g / L for ≥60 s and then drying it.

7. The method for extracting DNA from sorghum seeds according to claim 6, characterized in that, The sorghum seeds are sorghum seed coat, endosperm, and / or whole seeds.

8. The method for extracting DNA from sorghum seeds according to claim 6, characterized in that, The extraction method further includes using the extracted DNA as a DNA template to perform a PCR molecular reaction to amplify the DNA.

9. The method for extracting DNA from sorghum seeds according to claim 8, characterized in that, The PCR reaction system also includes PCR Master Mix, primers for the conserved sorghum sequence, and water; preferably, the nucleotide sequences of the primers for the conserved sorghum sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2; preferably, the PCR reaction conditions are: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 51.5℃ annealing for 30 s, 72℃ extension for 20 s, for 35 cycles; 72℃ complete extension for 10 min.

10. The use of the extract according to any one of claims 1 to 3, the kit according to claim 4, or the extraction method according to any one of claims 5 to 9 in the extraction of plant DNA.

Images