CN111254214A - Amplification method of sesame SSR molecular marker and application thereof - Google Patents

Abstract

The invention relates to an amplification method of sesame SSR molecular markers and application thereof in the technical field of biology, wherein the amplification method comprises the following steps: obtaining DNA of sesame; performing PCR amplification on the SSR molecular marker by taking the DNA of the sesame as a template; detecting a PCR amplification product by polyacrylamide gel electrophoresis; the DNA obtaining method of the sesame comprises the following steps: and (3) placing the fresh sesame leaves in 40 mu L of 0.5mol/L NaOH solution, standing for 10-15min, adding 400 mu L of 100mmol/L Tris-HCL solution with the pH of 8.0, and uniformly mixing to obtain the solution, namely the DNA solution of the sesame. The DNA template acquisition step is simple and convenient to operate, and a single sample only needs about 10 minutes; the required chemicals are few, only NaOH and Tris-HCL are needed, and the product is basically non-toxic and harmless; professional experimental equipment is not needed, and the cost is low.

Description

Amplification method of sesame SSR molecular marker and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an amplification method of sesame SSR molecular markers and application thereof.

Background

SSR (simple sequence repeats) markers, also called microsatellite DNA (Microsatelite DNA) markers, are one of the most widely used molecular markers in plant research in recent years, and have the advantages of high stability, rich information content, easy operation and the like, so the SSR markers are widely applied to aspects of genetic diversity, fingerprint identification, association analysis, genetic map construction, QTL positioning and the like. The traditional SSR molecular marker technology comprises the steps of DNA extraction, PCR amplification, gel electrophoresis detection and the like. In the process of DNA extraction, multiple steps of liquid nitrogen grinding, water bath, centrifugation, supernatant liquid suction, precipitation, rinsing, drying, dissolution and the like of a sample are required, the operation is complicated, a large amount of chemical reagents and related instruments and equipment are required, and the experimental process of large-scale samples is influenced to a certain extent. In addition, sesame contains a large amount of viscous biological substances such as polysaccharide and grease, and particularly in the process of extracting DNA from adult-stage leaves, the viscous substances seriously affect operations such as sample grinding, supernatant liquid suction, precipitation, airing, dissolution and the like in the process of extracting DNA, and the conditions of extraction failure or poor DNA quality often occur.

With the continuous and deep research of molecular marker detection and molecular marker-assisted selective breeding, the demand for large-scale development of SSR molecular marker operation will be greater and greater. Therefore, a simple amplification method of sesame SSR molecular markers is urgently needed to be established.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the defects of complex operation, high cost and poor DNA quality in the DNA extraction process in the prior art, so as to provide the amplification method of the sesame SSR molecular marker and the application thereof.

An amplification method of sesame SSR molecular markers comprises the following steps: extracting DNA of sesame; performing PCR amplification on the SSR molecular marker by taking the DNA of the sesame as a template;

the DNA extraction method of the sesame comprises the following steps: placing 0.3 mu g of fresh sesame leaves in 40 mu L of 0.5mol/L NaOH solution for standing for 10-15min, then adding 400 mu L of 100mmol/L Tris-HCL solution with pH of 8.0, and uniformly mixing to obtain a solution, namely the DNA solution of sesame;

the sesame leaf blade is selected from cotyledon at bud stage, full-spread leaf blade at seedling stage, four pairs of true leaf blade at seedling stage or upper young leaf blade at adult stage;

the diameter of the fresh leaves of the adopted sesame is 0.6-0.8 mm.

The reaction system of the PCR amplification is as follows: template 2. mu.l, 10 XTaq buffer with (NH)₄)₂SO₄1μl，25mmol Mgcl₂0.8. mu.l, 0.2. mu.l of 10mmol dNTP, 0.3. mu.l of 50 ng/. mu.l forward primer, 0.3. mu.l of 50 ng/. mu.l reverse primer, 0.2. mu.l of 5U/. mu.l Taq DNA polymerase, ddH₂O was supplemented to 10. mu.l.

The procedure of PCR amplification is as follows: denaturation at 94-95 deg.C for 3 min; 10 cycles: denaturation at 94 ℃ for 1min, annealing at 60-55 ℃ for 30s, decreasing by 0.5 ℃ per cycle, and extension at 72 ℃ for 45 s; 30 cycles: denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 45 s; finally, the mixture is stored at 72 ℃ for 10min and 4 ℃.

Polyacrylamide gel electrophoresis is carried out for 1.0-1.5h by 150-180V constant voltage electrophoresis;

the mass percentage concentration of the polyacrylamide gel is 6%;

the electrophoresis buffer is 1 XTBE solution.

The amplification method of the sesame SSR molecular marker is applied to hybrid identification or variety purity identification of sesame.

A method for identifying sesame hybrid seeds by using SSR molecular markers comprises the following steps:

adopting the simple amplification method to amplify the sesame F1 hybrid and the parents thereof with SSR molecular markers;

in the amplification product of the F1 hybrid, if there are bands specific to both male and female parents, it is a true hybrid, whereas it is a false hybrid.

A method for identifying sesame variety purity by SSR molecular markers comprises the following steps:

adopting the amplification method to amplify the sesame to be detected with the SSR molecular marker;

and if at least 1 SSR molecular marker locus different from other sesame individuals to be detected exists in a certain sesame individual to be detected, judging that the sesame individual to be detected is a hybrid strain.

The technical scheme of the invention has the following advantages:

1. the invention provides an amplification and detection method of sesame SSR molecular markers, which comprises the following steps: obtaining DNA of sesame; performing PCR amplification on the SSR molecular marker by taking the DNA of the sesame as a template; the DNA obtaining method of the sesame comprises the following steps: placing 0.3 mu g of fresh sesame leaves in 40 mu L of 0.5mol/L NaOH solution for standing for 10-15min, then adding 400 mu L of 100mmol/LpH 8.0.0 Tris-HCL solution, and uniformly mixing to obtain a solution, namely the DNA solution of sesame; the DNA obtaining method overcomes the influence of sticky substances in the sesame on the DNA extraction process, and can meet the requirement of the amplification of subsequent SSR molecular markers. The DNA template acquisition step is simple and convenient to operate, a very small amount of tissue samples are collected and then relevant reagents are added, only 10-15 minutes is needed for a single sample, compared with the traditional method, the method needs more than one day for grinding, water bath, centrifugation, precipitation, rinsing, drying, dissolving and other processes, and the efficiency is greatly improved; the method has the advantages that the required chemicals are few, only NaOH and Tris-HCL are needed, compared with the traditional CTAB method, the use of chemical reagents such as CTAB, chloroform, isoamylol, ethanol, acetone, EDTAD, liquid nitrogen and the like is reduced, and the method is basically non-toxic and harmless; professional experimental equipment such as a constant-temperature water bath kettle, a high-speed centrifuge, a low-temperature refrigerator, a fume hood and the like is not needed, and the cost is low; the leaves are selected as sample materials, the collection amount is very small, the detected plants are basically not damaged, and the normal growth of the plants after molecular detection is not influenced.

2. The PCR amplification program of the invention is as follows: denaturation at 94-95 deg.C for 3 min; 10 cycles: denaturation at 94 ℃ for 1min, annealing at 60-55 ℃ for 30s, decreasing by 0.5 ℃ per cycle, and extension at 72 ℃ for 45 s; 30 cycles: denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 45 s; finally, the DNA template is stored at the temperature of 72 ℃ for 10min and 4 ℃, and can be suitable for PCR amplification of a DNA template with lower content. An increase of 10 cycles was chosen: denaturation at 94 ℃ for 1min, annealing at 60-55 ℃ for 30s, decreasing by 0.5 ℃ in each cycle, and extending at 72 ℃ for 45s, so that the primer can better adapt to different primers, and the stability of SSR molecular marker amplification is ensured.

3. The invention provides the SSR molecular marker amplification and detection method suitable for sesame by simplifying the collection of tissue samples, quickly obtaining the DNA template of SSR molecular marker amplification, optimizing a PCR system and a PCR amplification program and establishing a matched gel detection technology.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the amplification results of example 1 of the present invention and comparative example 1; wherein, A is Burma black sesame, B is sesame strain Z11, C is Burma black sesame and Z11 hybrid strain F1; 1 is the amplification result of example 1, and 2 is the amplification result of comparative example 1; (1) (2) and (3) represent 3 pairs of different primers;

FIG. 2 shows the amplification results of example 2 of the present invention; wherein, A is the amplification result by using cotyledon sampling in the cotyledon full-expansion period, B is the amplification result by using four pairs of young and tender leaves in the true leaf period, and M is marker; 1.2 and 3 are 3 individuals, i.e. 3 replicates, respectively; (1) and (2) and (3) represent 3 different pairs of primers;

FIG. 3 shows the amplification results of example 3 of the present invention; wherein 1-14 are random 14 plant numbers in sesame strain Z11, and (1) - (5) represent 5 pairs of different primers.

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

Example 1

The materials used in this example were Burma black sesame in the four pairs of true leaf stages, strain Z11, F1 of Burma black sesame crossed with strain Z11. The steps of amplifying and detecting SSR molecular markers are as follows:

(1) obtaining DNA of sesame

Four young leaves at the true leaf stage were punched out using a 200. mu.l yellow gun head and sampled, approximately 0.6-0.8mm in diameter and approximately 0.3. mu.g in mass, into a 1.5ml centrifuge tube, and the samples on the black sesame burmese, strain Z11 and F1 plants were numbered A1, B1 and C1, respectively. Respectively adding 40 mul of 0.5mol/L NaOH solution into centrifuge tubes with the numbers of A1, B1 and C1, and standing for 10 min; then 400 mul of 100mmol/L Tris-HCl (pH 8.0) solution is respectively added into centrifuge tubes with the numbers of A1, B1 and C1, and the mixture is mixed evenly, the obtained solution is the DNA solution of the sesame, and the solution is preserved at 4 ℃ for standby.

(2) PCR amplification

And (3) amplifying the SSR molecular marker by using the DNA as a template.

The PCR reaction system (10. mu.l) was: DNA template 2. mu.l, 10 XTaq buffer with (NH)₄)₂SO₄1μl，25mmolMgCl₂0.8. mu.l, 0.2. mu.l of 10mmol dNTP, 0.3. mu.l of 50 ng/. mu.l forward primer, 0.3. mu.l of 50 ng/. mu.l reverse primer, 0.2. mu.l of 5U/. mu.l Taq DNA polymerase, ddH₂O 5.2μl。

Carrying out PCR amplification of SSR molecular markers by using a PCR instrument, wherein the amplification procedure is as follows: denaturation at 94 deg.C for 3 min; 10 cycles: denaturation at 94 ℃ for 1min, annealing at 60-55 ℃ for 30s, decreasing by 0.5 ℃ per cycle, and extension at 72 ℃ for 45 s; 30 cycles: denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 45 s; finally, the mixture is stored at 72 ℃ for 10min and 4 ℃.

The primers used were 3 pairs of SSR primers screened in bulk as shown in table 1.

Table 1 representative SSR primer sequences of 3 pairs used in example 1

(3) Electrophoretic detection

Carrying out electrophoretic separation on the PCR amplification product by using 6 mass percent polyacrylamide gel, wherein an electrophoretic buffer solution is a1 xTBE solution, the electrophoresis is carried out for 1.5h by adopting 150V constant voltage electrophoresis, after the electrophoresis is finished, fixing the gel by using 10 percent (volume percent) acetic acid solution for 10min, and then carrying out silver infiltration staining for 20min by using 0.2 percent (mass percent) silver nitrate aqueous solution; rinsing twice with distilled water for 2 minutes each time; then developing color in 1.5% (mass percent) sodium hydroxide and 0.4% (volume percent) formaldehyde mixed solution, finally rinsing the gel with clear water, photographing in a gel imaging system and storing data. The electrophoresis results are shown in FIG. 1.

Comparative example 1

Adopting a traditional SSR molecular marker operation method to carry out SSR molecular marker amplification and detection on the Myanmar black sesame, the strain Z11, the Myanmar black sesame and the strain Z11 hybridized F1 in the four pairs of true leaf stages, and comprising the following steps:

(1) DNA extraction of sesame by CTAB method

Respectively collecting 2-3 pieces of young leaves of Myanmar black sesame, strain Z11 and F1 plants in the true leaf period of four pairs of sesame, wherein the mass is about 2g, and the collected sample numbers are respectively marked as A2, B2 and C2; grinding a sample into powder by using liquid nitrogen, transferring the powder into a 2.0ml centrifuge tube, and correspondingly numbering; adding 800 μ l of 2 × CTAB extract, water-bathing at 65 deg.C for 45min, taking out, and cooling to room temperature (25 deg.C); adding 800. mu.l of a mixture of chloroform/isoamyl alcohol (v/v. 24/1), and slowly shaking for 5 min; centrifuging at 12000rpm for 15min, and collecting supernatant; adding 0.6 μ l of pre-cooled isopropanol at-20 deg.C, mixing, and standing for 15 min; centrifuging at 3000rpm for 3min, removing supernatant, leaving flocculent precipitate, adding 70% ethanol solution 1ml, and rinsing for 15 min; removing the upper layer solution, leaving flocculent precipitate, and air drying; after dissolving in 30. mu.l of TE buffer, the mixture was stored in a freezer at-20 ℃ until use.

The preparation method of the 2 xCTAB extracting solution comprises the following steps:

weighing 4g of hexadecyl trimethyl ammonium bromide (CTAB), 16.364g of NaCl, 20ml of 1M Tris-HCl (pH is 8.0) and 8ml of 0.5M EDTA (pH is 8.0), dissolving with 70ml of sterile ultrapure water, then fixing the volume to 200ml, sterilizing, cooling, adding 200 mu l of 2-mercaptoethanol (analytically pure), and shaking uniformly to obtain the product.

The preparation method of TE buffer solution is as follows:

1) preparation of 1M Tris-HCl (pH 8.0): weighing 6.06g of tris (hydroxymethyl) aminomethane, adding 40ml of ultrapure water for dissolving, dropwise adding concentrated HCl to adjust the pH value to 8.0, and fixing the volume to 50 ml;

2) formulation of 0.5M EDTA (pH 8.0): 9.306g of ethylene diamine tetraacetic acid disodium salt is weighed, 35ml of ultrapure water is added, vigorous stirring is carried out, the pH value is adjusted to 8.0 by NaOH, and the volume is fixed to 50 ml;

3) preparation of TE buffer: taking 1ml of the 1M Tris-HCl (pH 8.0) solution, 0.2ml of 0.5M EDTA (pH 8.0) solution and metering to 100ml with ultrapure water to obtain the final product.

(2) PCR amplification

And (3) amplifying the SSR molecular marker by using the DNA as a template.

The PCR reaction (15. mu.l) was: 2. mu.l of 50 ng/. mu.l template DNA, 1. mu.l of 50 ng/. mu.l forward primer, 1. mu.l of 50 ng/. mu.l reverse primer, 1.5. mu.l 10 XTaq buffer, 0.3. mu.l 10mMol dNTPs, 25mMol MgCl₂1.2. mu.l and 5U/. mu.l TaqDNA polymerase 0.2. mu.l; the primers used were 3 pairs of SSR primers in Table 1.

Carrying out PCR amplification of SSR molecular markers by using a PCR instrument, wherein the amplification procedure is as follows: denaturation at 94 deg.C for 3 min; 40 cycles: denaturation at 94 ℃ for 60s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 45 s; then, the extension was carried out at 72 ℃ for 10min, and the resulting product was stored at 4 ℃.

(3) Electrophoretic detection

Carrying out electrophoretic separation on the PCR amplification product by using 6 mass percent polyacrylamide gel, wherein an electrophoretic buffer solution is a1 xTBE solution, the electrophoresis is carried out for 1.5h by adopting 150V constant voltage electrophoresis, after the electrophoresis is finished, fixing the gel by using 10 percent (volume percent) acetic acid solution for 10min, and then carrying out silver infiltration staining for 20min by using 0.2 percent (mass percent) silver nitrate aqueous solution; rinsing twice with distilled water for 2 minutes each time; then developing color in 1.5% (mass percent) sodium hydroxide and 0.4% (volume percent) formaldehyde mixed solution, finally rinsing the gel with clear water, photographing in a gel imaging system and storing data. The electrophoresis results are shown in FIG. 1.

In FIG. 1, A is Myanmar black sesame, B is sesame strain Z11, and C is Myanmar black sesame and Z11 hybrid plant F1; 1 is the amplification result of example 1, and 2 is the amplification result of comparative example 1; (1) and (2) and (3) represent 3 different pairs of primers. As can be seen from the results in FIG. 1, the amplification results of the 3 pairs of primers using the method of the present invention are consistent with those of the conventional method, and it can be seen that the method of the present invention is as accurate as the SSR molecular marker amplification results of the conventional method.

The method for amplifying the SSR molecular marker in the example 1 can be used for identifying sesame hybrids, and the method in the example 1 is adopted to amplify the SSR molecular marker of the Myanmar black sesame, the strain Z11, the Myanmar black sesame and the strain Z11 hybridized F1; in the amplification product of the F1 hybrid, if there are bands specific to both male and female parents, it is a true hybrid, whereas it is a false hybrid. As can be seen from FIG. 1, the DNA bands of parents appeared in the F1 plants, indicating that F1 is a true hybrid.

Example 2

The material used in this example was sesame strain Z11, which was sampled at the cotyledon full-expansion stage and the four true-leaf stage. The steps of amplifying and detecting SSR molecular markers are as follows:

(1) obtaining DNA of sesame

Punching and sampling cotyledons in the full development stage of the cotyledons and four pairs of young leaves in the true leaf stage by using a 200-microliter yellow gun head, wherein the diameter of the sample is about 0.6-0.8mm, the mass of the sample is about 0.3 microgram, the sample is placed in a 1.5ml centrifuge tube, and each material is sampled for 3 times, and the sampling is regarded as 3 times of repetition, wherein the cotyledons in the full development stage of the cotyledons are numbered A1, A2 and A3, and the sampling numbers of the four pairs of young leaves in the true leaf stage are B1, B2 and B3. Respectively adding 40 μ L of 0.5mol/L NaOH solution into centrifuge tubes with numbers of A1, B1, C1, B1, B2 and B3, and standing for 10 min; then, 400 μ L of 100mmol/L Tris-HCl (PH 8.0) solution is respectively added into the centrifuge tube, and is uniformly mixed, the obtained solution is the DNA solution of the sesame, and the solution is preserved at 4 ℃ for standby.

(2) PCR amplification

And (3) amplifying the SSR molecular marker by using the DNA as a template.

The PCR reaction system (10. mu.l) was: DNA template 2. mu.l, 10 XTaq buffer with (NH)₄)₂SO₄1μl，25mmolMgCl₂0.8. mu.l, 0.2. mu.l of 10mmol dNTP, 0.3. mu.l of 50 ng/. mu.l forward primer, 0.3. mu.l of 50 ng/. mu.l reverse primer, 0.2. mu.l of 5U/. mu.l Taq DNA polymerase, ddH₂O 5.2μl。

Carrying out PCR amplification of SSR molecular markers by using a PCR instrument, wherein the amplification procedure is as follows: denaturation at 94 deg.C for 3 min; 10 cycles: denaturation at 94 ℃ for 1min, annealing at 60-55 ℃ for 30s, decreasing by 0.5 ℃ per cycle, and extension at 72 ℃ for 45 s; 30 cycles: denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 45 s; finally, the mixture is stored at 72 ℃ for 10min and 4 ℃.

The primers used were 3 pairs of SSR primers screened in bulk as shown in table 2.

Table 2 representative SSR primers used in example 2

(3) Electrophoretic detection

Carrying out electrophoretic separation on the PCR amplification product by using 6 mass percent polyacrylamide gel, wherein an electrophoretic buffer solution is a1 xTBE solution, the electrophoresis adopts 180V constant voltage electrophoresis for 1.0h, after the electrophoresis is finished, fixing the gel by using 10 volume percent of acetic acid solution for 10min, and then carrying out silver infiltration staining for 20min by using 0.2 mass percent of silver nitrate aqueous solution; rinsing twice with distilled water for 2 minutes each time; then developing color in 1.5% (mass percent) sodium hydroxide and 0.4% (volume percent) formaldehyde mixed solution, finally rinsing the gel with clear water, photographing in a gel imaging system and storing data. The electrophoresis results are shown in FIG. 2.

In FIG. 2, A is the amplification result obtained by sampling young leaves at the fully developed stage of cotyledons (cotyledons at the shoot stage), B is the amplification result obtained by sampling young leaves at the true leaf stage (leaves at the seedling stage), and M is marker; 1.2 and 3 are 3 individuals, i.e. 3 replicates, respectively; (1) and (2) and (3) represent 3 different pairs of primers. As can be seen from the results in FIG. 2, the amplification results of 3 pairs of primers are completely consistent, and it can be seen that the sample collection of the method of the present invention is suitable for the cotyledon at the bud stage of sesame and the young leaf at the seedling stage.

Example 3

The material used in this example was sesame line Z11 mixed with other sesame varieties, and the population was sampled at the cotyledon full-expansion stage. The SSR molecular marker is used for identifying the purity of the variety, and the specific operation steps are as follows:

(1) extraction of DNA of sesame

The cotyledons at the full-growth stage of the cotyledons are punched and sampled by a 200-microliter yellow gun head, the diameter of the sample is about 0.6-0.8mm, the mass of the sample is about 0.3 microgram, the sample is placed in a 1.5ml centrifuge tube, and 14 single plant cotyledon samples are randomly collected, and the numbers are 1, 2 … … 13 and 14. Respectively adding 40 mul of 0.5mol/L NaOH solution into 14 centrifuge tubes in which samples are put, and standing for 10 min; then adding 400 mul of 100mmol/L Tris-HCL (pH 8.0) solution into 14 centrifuge tubes respectively, mixing uniformly to obtain solution, namely the DNA solution of sesame, and storing at 4 ℃ for later use.

(2) PCR amplification

And (3) amplifying the SSR molecular marker by using the DNA as a template.

The PCR reaction system (10. mu.l) was: DNA template 2. mu.l, 10 XTaq buffer with (NH)₄)₂SO₄1μl，25mMolMgcl₂Mu.l, 10mMol dNTP 0.2. mu.l, 50 ng/. mu.l forward primer 0.3. mu.l, 50 ng/. mu.l reverse primer 0.3. mu.l, 5U/. mu.l Taq DNA polymerase 0.2. mu.l, ddH2O 5.2. mu.l.

Carrying out PCR amplification of SSR molecular markers by using a PCR instrument, wherein the amplification procedure is as follows: denaturation at 95 deg.C for 3 min; 10 cycles: denaturation at 94 ℃ for 1min, annealing at 60-55 ℃ for 30s, decreasing by 0.5 ℃ per cycle, and extension at 72 ℃ for 45 s; 30 cycles: denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 45 s; finally, the mixture is stored at 72 ℃ for 10min and 4 ℃.

The primers used were 5 pairs of SSR primers screened in bulk as shown in table 3.

Table 35 pairs of representative SSR primers used in example 3

(3) Electrophoretic detection

Carrying out electrophoretic separation on the PCR amplification product by using 6 mass percent polyacrylamide gel, wherein an electrophoretic buffer solution is a1 xTBE solution, the electrophoresis is carried out for 1.5h by adopting 150V constant voltage electrophoresis, after the electrophoresis is finished, fixing the gel by using 10 percent (volume percent) acetic acid solution for 10min, and then carrying out silver infiltration staining for 20min by using 0.2 percent (mass percent) silver nitrate aqueous solution; rinsing twice with distilled water for 2 minutes each time; then developing color in 1.5% (mass percent) sodium hydroxide and 0.4% (volume percent) formaldehyde mixed solution, finally rinsing the gel with clear water, photographing in a gel imaging system and storing data. The electrophoresis results are shown in FIG. 3.

In FIG. 3, 1 to 14 are random 14 plant numbers in sesame line Z11, and (1) to (5) represent 5 pairs of different primers. As can be seen from the results in FIG. 3, the amplification results of 5 pairs of primers all show that the plant with the number 2 is a hybrid plant, and is a non-strain Z11 plant, and it can be seen that the method is suitable for the identification of the purity of sesame varieties.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims (8)

1. An amplification method of sesame SSR molecular markers is characterized by comprising the following steps: obtaining DNA of sesame; performing PCR amplification on the SSR molecular marker by taking the DNA of the sesame as a template;

the DNA obtaining method of the sesame comprises the following steps: and (3) placing 0.3 mu g of fresh leaves in 40 mu L of 0.5mol/L NaOH solution, standing for 10-15min, adding 400 mu L of 100mmol/L Tris-HCL solution with the pH value of 8.0, and uniformly mixing to obtain the solution, namely the DNA solution of the sesame.

2. The amplification method according to claim 1, wherein the fresh leaves of sesame are selected from cotyledons at the shoot stage, full-spread leaves at the seedling stage, four pairs of leaves at the true leaf stage at the seedling stage, or upper young leaves at the adult stage;

the diameter of the sampled sesame leaf is 0.6-0.8 mm.

3. The amplification method according to claim 1 or 2, wherein the reaction system of the PCR amplification is: template 2. mu.l, 10 XTaq buffer with (NH)₄)₂SO₄1μl，25mmol MgCl₂Mu.l 0.8, 0.2. mu.l 10mmol dNTP, 0.3. mu.l 50 ng/. mu.l forward primer, 0.3. mu.l 50 ng/. mu.l reverse primer, 0.2. mu.l 5U/. mu.l Taq DNA polymerase, ddH₂O was supplemented to 10. mu.l.

4. The amplification method according to any one of claims 1 to 3, wherein the PCR amplification procedure is: denaturation at 94-95 deg.C for 3 min; 10 cycles: denaturation at 94 ℃ for 1min, annealing at 60-55 ℃ for 30s, decreasing by 0.5 ℃ per cycle, and extension at 72 ℃ for 45 s; 30 cycles: denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 45 s; finally, the mixture is stored at 72 ℃ for 10min and 4 ℃.

5. The amplification method according to any one of claims 1 to 4, further comprising a step of detecting the PCR amplification product, wherein the polyacrylamide gel electrophoresis is performed by 150-180V constant voltage electrophoresis for 1.0 to 1.5 hours;

the mass percentage concentration of the polyacrylamide gel is 6%;

the electrophoresis buffer is 1 XTBE solution.

6. Use of the method for amplifying the SSR molecular markers of sesame seeds according to any one of claims 1 to 5 for hybrid identification or variety purity identification of sesame seeds.

7. A method for identifying sesame hybrid seeds by utilizing SSR molecular markers is characterized by comprising the following steps:

performing SSR molecular marker amplification on sesame F1 hybrid and parents thereof by using the amplification method of any one of claims 1 to 5;

in the amplification product of the F1 hybrid, if there are bands specific to both male and female parents, it is a true hybrid, whereas it is a false hybrid.

8. A method for identifying sesame variety purity by SSR molecular markers is characterized by comprising the following steps:

carrying out SSR molecular marker amplification on sesame to be detected by adopting the amplification method of any one of claims 1 to 5;

and if at least 1 SSR molecular marker locus different from other sesame individuals to be detected exists in a certain sesame individual to be detected, judging that the sesame individual to be detected is a hybrid strain.

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