Method for identifying filial generations of plums by high-throughput SSR (simple sequence repeat) molecular marker technology

Abstract

The invention discloses a method for identifying a plum filial generation by a high-throughput SSR molecular marker technology, and relates to the technical field of molecular biology, the method for identifying the plum filial generation by the high-throughput SSR molecular marker comprises the following steps: preparing an SSR primer system, running an SSR-PCR amplification program, carrying out agarose gel electrophoresis and carrying out capillary electrophoresis, the SSR primer system comprises deionized water, dNTP, Buffer, F, R0, a DNA template and Taq enzyme, and a preparation method of the DNA template comprises the following steps: putting 78 original samples into a culture medium filled with antibiotics for culturing, adding chloramphenicol into the culture medium, suspending with normal saline, rinsing to remove residual culture medium, adding NaOH-SDS for cracking, adding potassium acetate to recover to be neutral, and then adding the Taq enzyme to obtain the DNA template. And separating impurities and DNA by using a centrifugal method, and purifying the DNA to obtain the DNA template. According to the method, the DNA template is designed, so that the function of rapidly extracting DNA in the identification process is realized, the problem that the identification efficiency is reduced due to long DNA extraction time in the identification process is solved, and the DNA extraction difficulty is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, in particular to a method for identifying plum hybrid offspring using a high-throughput SSR molecular marker technology. Background Art

[0002] Molecular identification technology refers to the use of polymorphisms at the DNA or RNA level of organisms for identification, genetic analysis and gene positioning. In a broad sense, it refers to heritable and detectable DNA sequences or proteins. In a narrow sense, it refers to specific DNA fragments that reflect differences between biological individuals or populations. It is based on nucleotide sequence variations in genetic material between individuals and is a direct feedback of genetic polymorphisms at the DNA level. The main types include molecular markers based on Southern hybridization, molecular markers based on repetitive sequences, and molecular markers based on PCR. Traditional SSR typing methods such as gel electrophoresis and capillary electrophoresis have low throughput and rely on the similarity of electrophoretic patterns for comparison, which is time-consuming and labor-intensive. And the accuracy is limited. With the development of high-throughput sequencing technology, SSR molecular marker technology has also achieved high-throughput and high-accuracy typing, providing a more efficient tool for genetic research. High-throughput SSR molecular marker technology is based on amplicon sequencing technology, which realizes typing at the single base level and can accurately type thousands of SSR sites of multiple varieties at the same time, reducing typing costs and time consumption, and improving typing accuracy. This technology avoids the comparison of electrophoresis patterns in traditional typing methods, and realizes direct comparison between any two varieties, greatly improving work efficiency. At the same time, its rich polymorphism and high amount of information make it have significant advantages in the fields of genetic breeding and gene positioning.

[0003] The development of SSR primers is difficult. The development and synthesis of new SSR primers requires high investment and high technical difficulty. There are insufficient existing SSR markers. The number of currently available SSR markers is limited and cannot cover all functional genes. This limits the application scope of SSR molecular marker technology to a certain extent. The detection of SSR polymorphism is limited. The detection and application of SSR polymorphism depends to a large extent on the effect of PCR amplification. Unsatisfactory PCR amplification will directly affect the application effect of SSR markers.

[0004] 1. Patent document CN104561332B discloses an SSR molecular marker for identifying the sex of Populus davidiana and its application. The above patent realizes that the identification primers are extremely specific and stable. There is no need for polyacrylamide gel electrophoresis fragment separation. Only conventional agarose gel electrophoresis detection is needed to complete the rapid identification of the sex of Populus davidiana, providing theoretical and technical guidance for the directional breeding of Populus davidiana. However, the above patent cannot realize the function of rapid DNA extraction during the identification process.

[0005] 2. Patent document CN111424108B discloses a method for identifying holly germplasm using SSR molecular markers. The above patent achieves a high polymorphism detection rate, better resolution, stability and reliability, simplicity and efficiency, but the above patent cannot achieve the function of removing protein impurities and RNA.

[0006] 3. Patent document CN108277295B discloses a group of corn SSR molecular markers suitable for capillary electrophoresis detection technology and their applications. The above patent can expand the range of available corn marker sites at the genome level; it provides new tools for the identification of corn varieties, germplasm resources, kinship evaluation, cytoplasmic genetic characteristics and other research, and has good application prospects. However, the above patent cannot achieve the function of denaturing DNA molecules.

[0007] 4. Patent document CN112029825B discloses the screening method and application of SSR molecular marker primers for cauliflower. The above patent has realized the construction of a method for rapid and accurate identification of cauliflower and broccoli varieties in my country, providing a theoretical basis for the future identification of seedling purity and authenticity and germplasm resource management in the industrialization of cauliflower. However, the above patent cannot achieve the functions of ensuring accurate detection and reducing costs and pollution.

[0008] In summary, the above patents cannot achieve the function of rapid DNA extraction during the identification process, cannot achieve the function of removing protein impurities and RNA, cannot achieve the function of denaturing DNA molecules, cannot achieve the function of ensuring detection accuracy and reducing costs and pollution, resulting in long DNA extraction time and reduced identification efficiency during the identification process, the impact of DNA impurities in the sample on the test results, DNA stability and low detection efficiency during capillary electrophoresis, low throughput, long time consumption, low accuracy when processing a large number of samples, and waste pollution to the environment;

[0009] To this end, the present application proposes a method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology that can achieve the functions of rapid DNA extraction during the identification process, removal of protein impurities and RNA, denaturation of DNA molecules, and ensuring accurate detection and reducing costs and pollution. Summary of the invention

[0010] The purpose of the present invention is to provide a method for identifying plum hybrid offspring using a high-throughput SSR molecular marker technology, so as to solve the technical problems proposed in the above-mentioned background technology that the functions of rapid DNA extraction during the identification process cannot be achieved, the function of removing protein impurities and RNA cannot be achieved, the function of denaturing DNA molecules cannot be achieved, and the function of ensuring accurate detection and reducing costs and pollution cannot be achieved, resulting in a long DNA extraction time during the identification process and reduced identification efficiency, the influence of DNA impurities in the sample on the detection results, DNA stability and low detection efficiency during capillary electrophoresis, low throughput, long time consumption, low accuracy when processing a large number of samples, and waste pollution to the environment.

[0011] To achieve the above object, the present invention provides the following technical solutions: a method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology, a method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology, the method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology comprises: preparing an SSR primer system, running an SSR-PCR amplification program, performing agarose gel electrophoresis and performing capillary electrophoresis, the SSR primer system comprises: deionized water, dNTP, Buffer, F, R0, a DNA template and Taq enzyme, and the preparation method of the DNA template is:

[0012] Step 1: 78 original samples were cultured in a culture medium containing antibiotics;

[0013] Step 2: Add chloramphenicol to the culture medium;

[0014] Step 3: Use physiological saline to suspend and rinse to remove residual culture medium;

[0015] Step 4: Add NaOH-SDS for lysis;

[0016] Step 5: After adding potassium acetate to restore neutrality, use centrifugation to separate impurities and DNA;

[0017] Step 6: Purify the DNA to obtain a DNA template.

[0018] Preferably, the method for preparing the DNA template also includes:

[0019] Step 1: prepare 1 mol / L NaCL solution, chloroform containing octanol, 0.15 mol / L NaCL solution, proteinase K, chloroform, anhydrous ethanol, 75% ethanol and deionized water;

[0020] Step 2: crush 78 samples in 0.15 mol / L NaCL solution;

[0021] Step 3: Add 1 mol / L NaCL solution for extraction to obtain DNP mucus;

[0022] Step 4: Add chloroform containing octanol and shake to emulsify;

[0023] Step 5: Centrifuge to remove protein, recover the aqueous phase, and precipitate the DNA sodium salt with 2 volumes of 95% ethanol;

[0024] Step 6: Wash the precipitate with 75% ethanol and dry it at room temperature;

[0025] Step 7: Add deionized water to dissolve the DNA and obtain a DNA template.

[0026] Preferably, the step of purifying DNA is:

[0027] Step 7: Prepare proteinase K, Tris-saturated phenol, chloroform, isoamyl alcohol, anhydrous ethanol, 70% ethanol, TE buffer, and RNase solution;

[0028] Step 8: Add proteinase K to the prepared DNA and place it on a shaker for digestion;

[0029] Step 9: Add an equal volume of Tris-saturated phenol, shake well and centrifuge to separate the upper DNA layer and transfer it to a new centrifuge tube;

[0030] Step 10: Add an equal volume of chloroform and isoamyl alcohol mixture to the supernatant, shake well and centrifuge to remove the upper aqueous phase;

[0031] Step 11: Add an equal volume of anhydrous ethanol to the supernatant, mix well and let stand;

[0032] Step 12: Separate the DNA, wash twice with 70% ethanol, and dry in a vacuum environment;

[0033] Step 13: Dissolve the dried DNA in TE buffer, add RNase solution for treatment, and extract with phenol / chloroform;

[0034] Step 14: Wash twice with 70% ethanol, dry in a vacuum environment, add TE buffer to dissolve the DNA, and prepare a DNA template.

[0035] Preferably, the method for identifying plum hybrid offspring is:

[0036] Step 1: Prepare the SSR primer system for PCR reaction system;

[0037] Step 2: Run the SSR-PCR amplification program; Step 3: Perform agarose gel electrophoresis on the products after the PCR reaction is completed;

[0038] Step 4: After the PCR reaction product is tested by agarose electrophoresis, it is tested by capillary electrophoresis;

[0039] Step 5: Analyze the capillary electrophoresis test results and draw conclusions.

[0040] Preferably, the capillary electrophoresis detection method is:

[0041] Step 6: Mix formamide and molecular weight internal standard at a volume ratio of 100:1, take 15 μl and add it to the sample plate, then add 1 μl of PCR product diluted 10 times;

[0042] Step 7: Use 3730XL sequencer to perform capillary electrophoresis on the samples;

[0043] Step 8: Use the Fragment fragment in Genemarker V2.2.0 software to analyze the initial data obtained by the sequencer;

[0044] Step 9: Compare the position of the molecular weight internal standard in each lane with the position of each sample peak to obtain the fragment size.

[0045] Preferably, the agarose gel electrophoresis detection is:

[0046] Step 10: Add TAE / TBE buffer to agarose powder and heat in a microwave until completely melted;

[0047] Step 11: Shake well and pour into the mold. After solidification, put the gel block into the electrophoresis tank;

[0048] Step 12: Add electrophoresis buffer to the electrophoresis tank until the liquid level is 2 mm above the gel surface;

[0049] Step 13: Extract the PCR reaction product for detection, mix the product with the loading buffer, and add it to the loading well;

[0050] Step 14: Connect the electrophoresis apparatus and electrophoresis tank, keep the voltage at 120V, and perform electrophoresis for 50 minutes;

[0051] Step 15: Stop electrophoresis, take out the gel block, stain it, and then observe and record the results.

[0052] Preferably, the SSR primer system has a total volume of 20 μl, including 14.8 μl of deionized water, 0.4 μl of dNTP, 2 μl of Buffer, 0.3 μl of F, 0.3 ul of R, 2 μl of DNA template and 0.2 μl of Taq enzyme.

[0053] Preferably, the formamide inhibits the formation of secondary structure of DNA molecules, thereby keeping the DNA in a single-stranded state during electrophoresis. Formamide reduces the interaction between nucleic acid molecules, thereby reducing the conformational changes of DNA and RNA during electrophoresis.

[0054] Preferably, the SSR-PCR amplification program is: pre-denaturation at 94°C for 5 min, deformation at 94°C for 30 s, annealing at 60°C for 45 s, and extension at 72°C for 50 s, which is one cycle, for a total of 35 cycles, and finally extension at 72°C for 5 min.

[0055] Preferably, the concentration of the agarose gel is 2.0%.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] 1. The present invention realizes the function of rapid DNA extraction during the identification process by designing a DNA template, solves the problem of long DNA extraction time and reduced identification efficiency during the identification process, can meet the processing of large-scale samples, improves experimental efficiency, reduces the difficulty of DNA extraction, and improves the accuracy of identification;

[0058] 2. The present invention is designed to purify DNA, thereby achieving the function of removing protein impurities and RNA, solving the problem of the influence of DNA impurities in the sample on the test results, reducing costs, improving the purity of DNA, and having relatively simple operation, improving the accuracy and reliability of the test, and reducing the identification time;

[0059] 3. The present invention realizes the function of denaturing DNA molecules by adding formamide, solves the problems of DNA stability and low detection efficiency during capillary electrophoresis, can denature DNA into a single-stranded state, improves the separation effect of electrophoresis and the accuracy of analysis, can stabilize RNA and denatured single-stranded DNA, and improves the stability of samples during electrophoresis;

[0060] 4. The present invention realizes the functions of ensuring accurate detection and reducing costs and pollution by designing agarose gel electrophoresis, solves the problems of low throughput, long time consumption, low accuracy and environmental pollution caused by waste when processing a large number of samples, can improve identification efficiency, improve the accuracy and reliability of identification results, simplify the operation process, reduce the difficulty of subsequent sample recovery, and reduce recovery costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a schematic diagram of the preparation of the DNA template of the present invention;

[0062] Figure 2 It is a schematic diagram of DNA purification of the present invention;

[0063] Figure 3 It is a schematic diagram of the identification method of the present invention;

[0064] Figure 4 Schematic diagram of capillary electrophoresis of the present invention;

[0065] Figure 5 It is a schematic diagram of agarose gel electrophoresis of the present invention;

[0066] Figure 6 It is a schematic diagram of the DNA template preparation of the present invention. DETAILED DESCRIPTION

[0067] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0068] Example 1

[0069] See also Figure 1 , Figure 2 and Figure 3 A method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology, the method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology comprises: preparing an SSR primer system, running an SSR-PCR amplification program, performing agarose gel electrophoresis and performing capillary electrophoresis, the SSR primer system comprises: deionized water, dNTP, Buffer, F, R0, a DNA template and Taq enzyme, and the preparation method of the DNA template is:

[0070] Step 1: 78 original samples were cultured in a culture medium containing antibiotics;

[0071] Step 2: Add chloramphenicol to the culture medium;

[0072] Step 3: Use physiological saline to suspend and rinse to remove residual culture medium;

[0073] Step 4: Add NaOH-SDS for lysis;

[0074] Step 5: After adding potassium acetate to restore neutrality, use centrifugation to separate impurities and DNA;

[0075] Step 6: Purify the DNA to obtain a DNA template;

[0076] The steps of purifying DNA are as follows:

[0077] Step 7: Prepare proteinase K, Tris-saturated phenol, chloroform, isoamyl alcohol, anhydrous ethanol, 70% ethanol, TE buffer, and RNase solution;

[0078] Step 8: Add proteinase K to the prepared DNA and place it on a shaker for digestion;

[0079] Step 9: Add an equal volume of Tris-saturated phenol, shake well and centrifuge to separate the upper DNA layer and transfer it to a new centrifuge tube;

[0080] Step 10: Add an equal volume of chloroform and isoamyl alcohol mixture to the supernatant, shake well and centrifuge to remove the upper aqueous phase;

[0081] Step 11: Add an equal volume of anhydrous ethanol to the supernatant, mix well and let stand;

[0082] Step 12: Separate the DNA, wash twice with 70% ethanol, and dry in a vacuum environment;

[0083] Step 13: Dissolve the dried DNA in TE buffer, add RNase solution for treatment, and extract with phenol / chloroform;

[0084] Step 14: Wash twice with 70% ethanol, dry in a vacuum environment, add TE buffer to dissolve the DNA, and prepare a DNA template;

[0085] The method for identifying the hybrid offspring of plum is as follows:

[0086] Step 1: Prepare the SSR primer system for PCR reaction system;

[0087] Step 2: Run the SSR-PCR amplification program; Step 3: Perform agarose gel electrophoresis on the products after the PCR reaction is completed;

[0088] Step 4: After the PCR reaction product is tested by agarose electrophoresis, it is tested by capillary electrophoresis;

[0089] Step 5: Analyze the capillary electrophoresis test results and draw conclusions;

[0090] Furthermore, 78 original samples were cultured in a culture medium containing antibiotics, chloramphenicol was added to the culture medium, suspended and rinsed with physiological saline to remove residual culture medium, lysed with NaOH-SDS, restored to neutrality by adding potassium acetate, and then separated from impurities and DNA by centrifugation. The DNA was purified to obtain a DNA template, thus realizing the function of rapid DNA extraction during the identification process and solving the problem of long DNA extraction time and reduced identification efficiency during the identification process. It can meet the processing of large-scale samples, improve experimental efficiency, reduce the difficulty of DNA extraction, and improve the accuracy of identification.

[0091] Example 2

[0092] See also Figure 2 and Figure 3 A method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology, the method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology comprises: preparing an SSR primer system, running an SSR-PCR amplification program, performing agarose gel electrophoresis and performing capillary electrophoresis, the SSR primer system comprises: deionized water, dNTP, Buffer, F, R0, a DNA template and Taq enzyme, and the preparation method of the DNA template is:

[0093] Step 1: 78 original samples were cultured in a culture medium containing antibiotics;

[0094] Step 2: Add chloramphenicol to the culture medium;

[0095] Step 3: Use physiological saline to suspend and rinse to remove residual culture medium;

[0096] Step 4: Add NaOH-SDS for lysis;

[0097] Step 5: After adding potassium acetate to restore neutrality, use centrifugation to separate impurities and DNA;

[0098] Step 6: Purify the DNA to obtain a DNA template;

[0099] The steps of purifying DNA are as follows:

[0100] Step 7: Prepare proteinase K, Tris-saturated phenol, chloroform, isoamyl alcohol, anhydrous ethanol, 70% ethanol, TE buffer, and RNase solution;

[0101] Step 8: Add proteinase K to the prepared DNA and place it on a shaker for digestion;

[0102] Step 9: Add an equal volume of Tris-saturated phenol, shake well and centrifuge to separate the upper DNA layer and transfer it to a new centrifuge tube;

[0103] Step 10: Add an equal volume of chloroform and isoamyl alcohol mixture to the supernatant, shake well and centrifuge to remove the upper aqueous phase;

[0104] Step 11: Add an equal volume of anhydrous ethanol to the supernatant, mix well and let stand;

[0105] Step 12: Separate the DNA, wash twice with 70% ethanol, and dry in a vacuum environment;

[0106] Step 13: Dissolve the dried DNA in TE buffer, add RNase solution for treatment, and extract with phenol / chloroform;

[0107] Step 14: Wash twice with 70% ethanol, dry in a vacuum environment, add TE buffer to dissolve the DNA, and prepare a DNA template;

[0108] The method for identifying the hybrid offspring of plum is as follows:

[0109] Step 1: Prepare the SSR primer system for PCR reaction system;

[0110] Step 2: Run the SSR-PCR amplification program; Step 3: Perform agarose gel electrophoresis on the products after the PCR reaction is completed;

[0111] Step 4: After the PCR reaction product is tested by agarose electrophoresis, it is tested by capillary electrophoresis;

[0112] Step 5: Analyze the capillary electrophoresis test results and draw conclusions;

[0113] Further, proteinase K, Tris-saturated phenol, chloroform, isoamyl alcohol, anhydrous ethanol, 70% ethanol, TE buffer and RNase solution are prepared, proteinase K is added to the prepared DNA, and the mixture is placed in a shaker for digestion, an equal volume of Tris-saturated phenol is added, the mixture is shaken and centrifuged, the upper DNA layer is separated and transferred to a new centrifuge tube, an equal volume of a mixture of chloroform and isoamyl alcohol is added to the supernatant, the mixture is shaken and centrifuged, the upper aqueous phase is removed, an equal volume of anhydrous ethanol is added to the supernatant, the mixture is mixed and allowed to stand, the DNA is separated, the DNA is washed twice with 70% ethanol, and the mixture is dried in a vacuum environment, the dried DNA is dissolved in TE buffer, the RNase solution is added for treatment, phenol / chloroform is used for extraction, the DNA is washed twice with 70% ethanol, and the mixture is dried in a vacuum environment, TE buffer is added to dissolve the DNA, and a DNA template is prepared, thereby realizing the function of removing protein impurities and RNA, solving the problem of the influence of DNA impurities in the sample on the test results, reducing costs, and improving the purity of DNA, and the operation is relatively simple, improving the accuracy and reliability of detection, and reducing the identification time.

[0114] Example 3

[0115] See also Figure 3 and Figure 4 , a method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology, the method for identifying plum hybrid offspring is:

[0116] Step 1: Prepare the SSR primer system for PCR reaction system;

[0117] Step 2: Run the SSR-PCR amplification program; Step 3: Perform agarose gel electrophoresis on the products after the PCR reaction is completed;

[0118] Step 4: After the PCR reaction product is tested by agarose electrophoresis, it is tested by capillary electrophoresis;

[0119] Step 5: Analyze the capillary electrophoresis test results and draw conclusions;

[0120] The method of capillary electrophoresis detection is:

[0121] Step 6: Mix formamide and molecular weight internal standard at a volume ratio of 100:1, take 15 μl and add it to the sample plate, then add 1 μl of PCR product diluted 10 times;

[0122] Step 7: Use 3730XL sequencer to perform capillary electrophoresis on the samples;

[0123] Step 8: Use the Fragment fragment in Genemarker V2.2.0 software to analyze the initial data obtained by the sequencer;

[0124] Step 9: Compare the position of the molecular weight internal standard in each lane with the position of each sample peak to obtain the fragment size;

[0125] The formamide inhibits the formation of secondary structures of DNA molecules, thereby keeping the DNA in a single-stranded state during electrophoresis. The formamide reduces the interaction between nucleic acid molecules, thereby reducing the conformational changes of DNA and RNA during electrophoresis.

[0126] Further, formamide and the molecular weight internal standard were mixed at a volume ratio of 100:1, 15 μl was taken and added to the sample plate, and then 1 μl of the PCR product diluted 10 times was added, and the sample was detected by capillary electrophoresis using a 3730XL sequencer. The initial data obtained by the sequencer was analyzed using the Fragment fragment in the Genemarker V2.2.0 software, and the position of the molecular weight internal standard in each lane was compared with the position of the peak of each sample to obtain the fragment size. Formamide inhibits the formation of the secondary structure of the DNA molecule, so that the DNA remains in a single-stranded state during electrophoresis. Formamide reduces the interaction between nucleic acid molecules and reduces the conformational changes of DNA and RNA during electrophoresis, thereby achieving the function of denaturing DNA molecules, solving the problems of DNA stability and low detection efficiency during capillary electrophoresis, and can denature DNA into a single-stranded state, thereby improving the separation effect of electrophoresis and the accuracy of analysis, and can stabilize RNA and denatured single-stranded DNA, thereby improving the stability of the sample during electrophoresis.

[0127] Example 4

[0128] See also Figure 3 and Figure 5 , a method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology, the method for identifying plum hybrid offspring is:

[0129] Step 1: Prepare the SSR primer system for PCR reaction system;

[0130] Step 2: Run the SSR-PCR amplification program; Step 3: Perform agarose gel electrophoresis on the products after the PCR reaction is completed;

[0131] Step 4: After the PCR reaction product is tested by agarose electrophoresis, it is tested by capillary electrophoresis;

[0132] Step 5: Analyze the capillary electrophoresis test results and draw conclusions;

[0133] The agarose gel electrophoresis detection is:

[0134] Step 10: Add TAE / TBE buffer to agarose powder and heat in a microwave until completely melted;

[0135] Step 11: Shake well and pour into the mold. After solidification, put the gel block into the electrophoresis tank;

[0136] Step 12: Add electrophoresis buffer to the electrophoresis tank until the liquid level is 2 mm above the gel surface;

[0137] Step 13: Extract the PCR reaction product for detection, mix the product with the loading buffer, and add it to the loading well;

[0138] Step 14: Connect the electrophoresis apparatus and electrophoresis tank, keep the voltage at 120V, and perform electrophoresis for 50 minutes;

[0139] Step 15: Stop the electrophoresis, take out the gel block, stain it, and then observe and record the results;

[0140] The concentration of the agarose gel is 2.0%;

[0141] Further, TAE / TBE buffer is added to agarose powder, the mixture is placed in a microwave oven and heated until completely melted, the mixture is shaken and poured into a mold, and after solidification, the gel block is placed in an electrophoresis tank, and electrophoresis buffer is added to the electrophoresis tank until the liquid level is 2 mm higher than the gel surface, PCR reaction products are extracted for detection, the products are mixed with loading buffer, added to the loading well, the electrophoresis instrument and the electrophoresis tank are connected, the voltage is maintained at 120 V, electrophoresis is performed for 50 minutes, the electrophoresis is stopped, the gel block is taken out, and the results are observed and recorded after staining. The concentration of agarose gel is 2.0%, which realizes the functions of ensuring accurate detection and reducing costs and pollution, solves the problems of low throughput, long time consumption, low accuracy and environmental pollution caused by waste when processing a large number of samples, can improve identification efficiency, improve the accuracy and reliability of identification results, simplify the operation process, reduce the difficulty of subsequent sample recovery, and reduce the recovery cost.

[0142] Example 5

[0143] See also Figure 1 , Figure 2 , Figure 3 , Figure 4 and Figure 5 A method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology, the method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology comprises: preparing an SSR primer system, running an SSR-PCR amplification program, performing agarose gel electrophoresis and performing capillary electrophoresis, the SSR primer system comprises: deionized water, dNTP, Buffer, F, R0, a DNA template and Taq enzyme, and the preparation method of the DNA template is:

[0144] Step 1: 78 original samples were cultured in a culture medium containing antibiotics;

[0145] Step 2: Add chloramphenicol to the culture medium;

[0146] Step 3: Use physiological saline to suspend and rinse to remove residual culture medium;

[0147] Step 4: Add NaOH-SDS for lysis;

[0148] Step 5: After adding potassium acetate to restore neutrality, use centrifugation to separate impurities and DNA;

[0149] Step 6: Purify the DNA to obtain a DNA template;

[0150] The steps of purifying DNA are as follows:

[0151] Step 7: Prepare proteinase K, Tris-saturated phenol, chloroform, isoamyl alcohol, anhydrous ethanol, 70% ethanol, TE buffer, and RNase solution;

[0152] Step 8: Add proteinase K to the prepared DNA and place it on a shaker for digestion;

[0153] Step 9: Add an equal volume of Tris-saturated phenol, shake well and centrifuge to separate the upper DNA layer and transfer it to a new centrifuge tube;

[0154] Step 10: Add an equal volume of chloroform and isoamyl alcohol mixture to the supernatant, shake well and centrifuge to remove the upper aqueous phase;

[0155] Step 11: Add an equal volume of anhydrous ethanol to the supernatant, mix well and let stand;

[0156] Step 12: Separate the DNA, wash twice with 70% ethanol, and dry in a vacuum environment;

[0157] Step 13: Dissolve the dried DNA in TE buffer, add RNase solution for treatment, and extract with phenol / chloroform;

[0158] Step 14: Wash twice with 70% ethanol, dry in a vacuum environment, add TE buffer to dissolve the DNA, and prepare a DNA template;

[0159] The method for identifying the hybrid offspring of plum is as follows:

[0160] Step 1: Prepare the SSR primer system for PCR reaction system;

[0161] Step 2: Run the SSR-PCR amplification program; Step 3: Perform agarose gel electrophoresis on the products after the PCR reaction is completed;

[0162] Step 4: After the PCR reaction product is tested by agarose electrophoresis, it is tested by capillary electrophoresis;

[0163] Step 5: Analyze the capillary electrophoresis test results and draw conclusions;

[0164] The method of capillary electrophoresis detection is:

[0165] Step 6: Mix formamide and molecular weight internal standard at a volume ratio of 100:1, take 15 μl and add it to the sample plate, then add 1 μl of PCR product diluted 10 times;

[0166] Step 7: Use 3730XL sequencer to perform capillary electrophoresis on the samples;

[0167] Step 8: Use the Fragment fragment in Genemarker V2.2.0 software to analyze the initial data obtained by the sequencer;

[0168] Step 9: Compare the position of the molecular weight internal standard in each lane with the position of each sample peak to obtain the fragment size;

[0169] The agarose gel electrophoresis detection is:

[0170] Step 10: Add TAE / TBE buffer to agarose powder and heat in a microwave until completely melted;

[0171] Step 11: Shake well and pour into the mold. After solidification, put the gel block into the electrophoresis tank;

[0172] Step 12: Add electrophoresis buffer to the electrophoresis tank until the liquid level is 2 mm above the gel surface;

[0173] Step 13: Extract the PCR reaction product for detection, mix the product with the loading buffer, and add it to the loading well;

[0174] Step 14: Connect the electrophoresis apparatus and electrophoresis tank, keep the voltage at 120V, and perform electrophoresis for 50 minutes;

[0175] Step 15: Stop the electrophoresis, take out the gel block, stain it, and then observe and record the results;

[0176] The SSR primer system contains 20 μl in total, including 14.8 μl of deionized water, 0.4 μl of dNTP, 2 μl of Buffer, 0.3 μl of F, 0.3 ul of R, 2 μl of DNA template and 0.2 μl of Taq enzyme;

[0177] The formamide inhibits the formation of secondary structures of DNA molecules, thereby keeping the DNA in a single-stranded state during electrophoresis. The formamide reduces the interaction between nucleic acid molecules, thereby reducing the conformational changes of DNA and RNA during electrophoresis.

[0178] The SSR-PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 60°C for 45 s, and extension at 72°C for 50 s, for one cycle, for a total of 35 cycles, and finally extension at 72°C for 5 min;

[0179] The concentration of the agarose gel is 2.0%;

[0180] Furthermore, an SSR primer system for the PCR reaction system was prepared, with a total of 20 μl of the SSR primer system, including 14.8 μl of deionized water, 0.4 μl of dNTP, 2 μl of Buffer, 0.3 μl of F, 0.3 ul of R, 2 μl of DNA template and 0.2 μl of Taq enzyme. The SSR-PCR amplification program was performed as follows: pre-denaturation at 94°C for 5 min, deformation at 94°C for 30 s, renaturation at 60°C for 45 s, and extension at 72°C for 50 s, which was one cycle. There were 35 cycles in total, and the final extension was performed at 72°C for 5 min. The products after the completion of the PCR reaction were detected by agarose gel electrophoresis. The products after the PCR reaction were detected by capillary electrophoresis after passing the agarose electrophoresis random inspection. The capillary electrophoresis detection results were analyzed, and it was concluded that by identifying the purified DNA, the function of rapid and accurate identification was achieved, the problems of low identification efficiency and inaccurate identification results were solved, the influence of protein and RNA on the identification process was avoided, and the reliability of the identification results was improved.

[0181] Example 6

[0182] See also Figure 3 , Figure 4 , Figure 5 and Figure 6 , a method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology, and the preparation method of the DNA template also includes:

[0183] Step 1: prepare 1 mol / L NaCL solution, chloroform containing octanol, 0.15 mol / L NaCL solution, proteinase K, chloroform, anhydrous ethanol, 75% ethanol and deionized water;

[0184] Step 2: crush 78 samples in 0.15 mol / L NaCL solution;

[0185] Step 3: Add 1 mol / L NaCL solution for extraction to obtain DNP mucus;

[0186] Step 4: Add chloroform containing octanol and shake to emulsify;

[0187] Step 5: Centrifuge to remove protein, recover the aqueous phase, and precipitate the DNA sodium salt with 2 volumes of 95% ethanol;

[0188] Step 6: Wash the precipitate with 75% ethanol and dry it at room temperature;

[0189] Step 7: Add deionized water to dissolve the DNA to obtain a DNA template;

[0190] The method for identifying the hybrid offspring of plum is as follows:

[0191] Step 1: Prepare the SSR primer system for PCR reaction system;

[0192] Step 2: Run the SSR-PCR amplification program; Step 3: Perform agarose gel electrophoresis on the products after the PCR reaction is completed;

[0193] Step 4: After the PCR reaction product is tested by agarose electrophoresis, it is tested by capillary electrophoresis;

[0194] Step 5: Analyze the capillary electrophoresis test results and draw conclusions;

[0195] The method of capillary electrophoresis detection is:

[0196] Step 6: Mix formamide and molecular weight internal standard at a volume ratio of 100:1, take 15 μl and add it to the sample plate, then add 1 μl of PCR product diluted 10 times;

[0197] Step 7: Use 3730XL sequencer to perform capillary electrophoresis on the samples;

[0198] Step 8: Use the Fragment fragment in Genemarker V2.2.0 software to analyze the initial data obtained by the sequencer;

[0199] Step 9: Compare the position of the molecular weight internal standard in each lane with the position of each sample peak to obtain the fragment size;

[0200] The agarose gel electrophoresis detection is:

[0201] Step 10: Add TAE / TBE buffer to agarose powder and heat in a microwave until completely melted;

[0202] Step 11: Shake well and pour into the mold. After solidification, put the gel block into the electrophoresis tank;

[0203] Step 12: Add electrophoresis buffer to the electrophoresis tank until the liquid level is 2 mm above the gel surface;

[0204] Step 13: Extract the PCR reaction product for detection, mix the product with the loading buffer, and add it to the loading well;

[0205] Step 14: Connect the electrophoresis apparatus and electrophoresis tank, keep the voltage at 120V, and perform electrophoresis for 50 minutes;

[0206] Step 15: Stop the electrophoresis, take out the gel block, stain it, and then observe and record the results;

[0207] The SSR primer system contains 20 μl in total, including 14.8 μl of deionized water, 0.4 μl of dNTP, 2 μl of Buffer, 0.3 μl of F, 0.3 ul of R, 2 μl of DNA template and 0.2 μl of Taq enzyme;

[0208] The formamide inhibits the formation of secondary structures of DNA molecules, thereby keeping the DNA in a single-stranded state during electrophoresis. The formamide reduces the interaction between nucleic acid molecules, thereby reducing the conformational changes of DNA and RNA during electrophoresis.

[0209] The SSR-PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 60°C for 45 s, and extension at 72°C for 50 s, for one cycle, for a total of 35 cycles, and finally extension at 72°C for 5 min;

[0210] The concentration of the agarose gel is 2.0%;

[0211] Further, 1 mol / L NaCL solution, chloroform containing octanol, 0.15 mol / L NaCL solution, proteinase K, chloroform, anhydrous ethanol, 75% ethanol and deionized water are prepared, 78 samples are crushed in 0.15 mol / L NaCL solution, 1 mol / L NaCL solution is added for extraction to obtain DNP mucus, chloroform containing octanol is added for shaking and emulsification, protein is removed by centrifugation, an aqueous phase is recovered, DNA sodium salt is precipitated with 2 volumes of 95% ethanol, the precipitate is washed with 75% ethanol, dried at room temperature, deionized water is added to dissolve DNA to obtain a DNA template, and the prepared DNA template is used for subsequent identification operations, thereby realizing the functions of rapid DNA extraction and purification, solving the problems of complicated DNA extraction operations and high costs, reducing costs, accelerating identification speed, and increasing the accuracy and reliability of identification results.

[0212] Working principle: Under alkaline conditions, the double helix structure of linear chromosome DNA is untied and denatured. Although the hydrogen bonds of plasmid DNA are broken, the two complementary chains are entangled with each other and tightly bound together. When potassium acetate is added to restore to neutrality, the chromosome DNA molecules are difficult to renature, while the plasmid DNA molecules renature quickly. During centrifugation, the chromosome DNA is precipitated together with the cell fragments, and the plasmid DNA remains in the supernatant. After precipitation with ethanol, a relatively pure plasmid DNA is obtained.

[0213] Organic solvents such as proteinase K, Tris-saturated phenol, chloroform and RNase solution are added to relatively pure plasmid DNA, impurities such as protein, polysaccharide, phenol and RNA are removed by extraction with organic solvents, washed with anhydrous ethanol, dried in a vacuum environment, and the purified plasmid DNA is dissolved in TE buffer to obtain a DNA template;

[0214] Formamide destroys the hydrogen bonds in DNA molecules, converting the double-stranded structure of DNA molecules into a single-stranded structure, reducing the interaction between DNA molecules, stabilizing RNA and denatured single-stranded DNA, and facilitating sample injection and separation. At the same time, adding formamide to the buffer solution can inhibit electroosmotic flow and improve separation efficiency.

[0215] Agarose gel electrophoresis is based on the fact that the migration speed of molecules in the gel under the action of an electric field is related to their molecular size and the amount of net charge they carry. When molecules are placed in an electric field, they will move to the appropriate motor at a certain speed, which is proportional to the electric field strength and the net charge carried by the molecules. Agarose gel has a network structure, and material molecules will encounter resistance when passing through. Large molecules will encounter greater resistance when surging. The separation of charged particles depends not only on the nature and amount of the net charge, but also on the molecular size.

[0216] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology, characterized in that: The method for identifying plum hybrid offspring by high-throughput SSR molecular markers comprises: preparing an SSR primer system, running an SSR-PCR amplification program, performing agarose gel electrophoresis and performing capillary electrophoresis, wherein the SSR primer system comprises: deionized water, dNTP, Buffer, F, R0, a DNA template and Taq enzyme, and the preparation method of the DNA template is: Step 1: 78 original samples were cultured in a culture medium containing antibiotics; Step 2: Add chloramphenicol to the culture medium; Step 3: Use physiological saline to suspend and rinse to remove residual culture medium; Step 4: Add NaOH-SDS for lysis; Step 5: After adding potassium acetate to restore neutrality, use centrifugation to separate impurities and DNA; Step 6: Purify the DNA to obtain a DNA template.

2. The method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology according to claim 1, characterized in that: The preparation method of the DNA template also includes: Step 1: prepare 1 mol / L NaCL solution, chloroform containing octanol, 0.15 mol / L NaCL solution, proteinase K, chloroform, anhydrous ethanol, 75% ethanol and deionized water; Step 2: crush 78 samples in 0.15 mol / L NaCL solution; Step 3: Add 1 mol / L NaCL solution for extraction to obtain DNP mucus; Step 4: Add chloroform containing octanol and shake to emulsify; Step 5: Centrifuge to remove protein, recover the aqueous phase, and precipitate the DNA sodium salt with 2 volumes of 95% ethanol; Step 6: Wash the precipitate with 75% ethanol and dry it at room temperature; Step 7: Add deionized water to dissolve the DNA and obtain a DNA template.

3. The method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology according to claim 1, characterized in that: The steps of purifying DNA are as follows: Step 7: Prepare proteinase K, Tris-saturated phenol, chloroform, isoamyl alcohol, anhydrous ethanol, 70% ethanol, TE buffer, and RNase solution; Step 8: Add proteinase K to the prepared DNA and place it on a shaker for digestion; Step 9: Add an equal volume of Tris-saturated phenol, shake well and centrifuge to separate the upper DNA layer and transfer it to a new centrifuge tube; Step 10: Add an equal volume of chloroform and isoamyl alcohol mixture to the supernatant, shake well and centrifuge to remove the upper aqueous phase; Step 11: Add an equal volume of anhydrous ethanol to the supernatant, mix well and let stand; Step 12: Separate the DNA, wash twice with 70% ethanol, and dry in a vacuum environment; Step 13: Dissolve the dried DNA in TE buffer, add RNase solution for treatment, and extract with phenol / chloroform; Step 14: Wash twice with 70% ethanol, dry in a vacuum environment, add TE buffer to dissolve the DNA, and prepare a DNA template.

4. The method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology according to claim 1, characterized in that: The method for identifying the hybrid offspring of plum is as follows: Step 1: Prepare the SSR primer system for PCR reaction system; Step 2: Run the SSR-PCR amplification program; Step 3: Perform agarose gel electrophoresis on the products after the PCR reaction is completed; Step 4: After the PCR reaction product is tested by agarose electrophoresis, it is tested by capillary electrophoresis; Step 5: Analyze the capillary electrophoresis test results and draw conclusions.

5. The method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology according to claim 1, characterized in that: The capillary electrophoresis detection method is: Step 6: Mix formamide and molecular weight internal standard at a volume ratio of 100:1, take 15 μl and add it to the sample plate, then add 1 μl of PCR product diluted 10 times; Step 7: Use 3730XL sequencer to perform capillary electrophoresis on the samples; Step 8: Use the Fragment fragment in Genemarker V2.2.0 software to analyze the initial data obtained by the sequencer; Step 9: Compare the position of the molecular weight internal standard in each lane with the position of each sample peak to obtain the fragment size.

6. The method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology according to claim 1, characterized in that: The agarose gel electrophoresis detection is: Step 10: Add TAE / TBE buffer to agarose powder and heat in a microwave until completely melted; Step 11: Shake well and pour into the mold. After solidification, put the gel block into the electrophoresis tank; Step 12: Add electrophoresis buffer to the electrophoresis tank until the liquid level is 2 mm above the gel surface; Step 13: Extract the PCR reaction product for detection, mix the product with the loading buffer, and add it to the loading well; Step 14: Connect the electrophoresis apparatus and electrophoresis tank, keep the voltage at 120V, and perform electrophoresis for 50 minutes; Step 15: Stop electrophoresis, take out the gel block, stain it, and then observe and record the results.

7. The method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology according to claim 1, characterized in that: The SSR primer system has a total volume of 20 μl, including 14.8 μl of deionized water, 0.4 μl of dNTP, 2 μl of Buffer, 0.3 μl of F, 0.3 ul of R, 2 μl of DNA template and 0.2 μl of Taq enzyme.

8. The method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology according to claim 5, characterized in that: The formamide inhibits the formation of the secondary structure of the DNA molecule, thereby keeping the DNA in a single-stranded state during the electrophoresis process. The formamide reduces the interaction between nucleic acid molecules, thereby reducing the conformational changes of DNA and RNA during the electrophoresis process.

9. The method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology according to claim 1, characterized in that: The SSR-PCR amplification program is: pre-denaturation at 94°C for 5 min, deformation at 94°C for 30 s, annealing at 60°C for 45 s, and extension at 72°C for 50 s, which is one cycle, for a total of 35 cycles, and finally extension at 72°C for 5 min.

10. The method for identifying plum hybrid offspring using high-throughput SSR molecular marker technology according to claim 6, characterized in that: The concentration of the agarose gel was 2.0%.

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