CN110541044A - Molecular marker primer combination for identifying peach fruit nuclear separation character and application thereof - Google Patents
Molecular marker primer combination for identifying peach fruit nuclear separation character and application thereof Download PDFInfo
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- 240000006413 Prunus persica var. persica Species 0.000 title 1
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Abstract
The invention discloses a group of molecular marker primer combinations capable of identifying peach germplasm nuclear-sticking character and application thereof, which are expected to be applied to peach nuclear-sticking character molecular marker-assisted selection. The invention provides a group of molecular marker primer combinations capable of identifying peach kernel sticking and separating characters, which are any one of JYSNP-1, JYSNP-2 and JYSNP-3 and can be used for quickly identifying the peach kernel sticking and separating characters of breeding materials. The method for assisting in screening the excellent peach germplasm resources based on the developed molecular marker primer combination has important guiding significance on the prediction of the peach sticky nuclear character phenotype, and has the advantages of simplicity in operation, high efficiency, rapidness, low cost, accurate result and the like.
Description
Technical Field
the invention relates to a group of molecular marker primer combinations for identifying the nuclear separation character of peach fruits and application thereof, belonging to the field of molecular biology.
Background
Peach [ Prunus persica (L.) Batsch ] belongs to Rosaceae (Rosaceae), Prunus (Prunus L.), originates from western region of China, and is one of important fruit trees cultivated in China. China has abundant peach germplasm resources, and the breeding of new superior varieties with higher economic value by utilizing the existing resources is one of the effective ways for improving the quality and the yield of the peaches in China. The sticking and the separation of the pulp from the pit are one of the important characters of the peach fruit; the core separation means that when a person eats or breaks the fruits, the pulp can be smoothly separated from the peach core, and no pulp remains on the peach core; the sticky core means that the pulp cannot be separated from the peach core smoothly, and a lot of pulp remains on the peach core. In the fruit tree germplasm resource descriptor, the peach sticky nuclear-abscission trait is classified into 3 grades of nuclear abscission, half nuclear abscission and nuclear adhesion.
the method for breeding the excellent variety by the conventional crossbreeding method has the defects of long breeding period, large floor area, high cultivation management cost and the like. The development of modern biotechnology provides an effective auxiliary method for peach breeding, namely DNA molecular markers, such as SSR, AFLP, SNP and the like, directly take genetic material DNA as research objects to reveal genetic diversity among species, so that early identification and selection work in the breeding process can be effectively carried out. It is believed that molecular marker assisted breeding will play a greater role in the near future with the further development of molecular biotechnology and the increasing trend of various crop maps.
in recent years, although molecular markers such as SSR and AFLP linked with peach fruit nuclear-attached genes are developed in related researches, certain linkage distance exists, the influence of a plurality of factors such as marker density, quantity and research means is limited, reference is provided for the research of auxiliary breeding of the molecular markers for peach nuclear-attached traits, and the practical value, the accuracy and the detection efficiency are unknown. The invention patent with the publication number of CN201511000523.7 discloses a gene for regulating and controlling the softening character of a nuclear isolated peach fruit and application thereof, wherein a molecular marker associated with the character of the nuclear isolated fruit is provided, and the gene can be used for detecting the phenotype of the nuclear isolated fruit; however, the molecular marker PCR amplification product fragment is too long (>1000bp), and is difficult to be applied to high-throughput detection technology platforms such as fluorescence capillary electrophoresis and fluorescence quantitative PCR instrument genotyping (usually, the product fragment length is required to be less than 500bp, but the product fragment length is recommended to be less than 200bp, and the effect is good), in addition, the molecular marker is small in quantity, a sample needs to be detected one by one, high-throughput typing cannot be realized, and when the task of detecting a large number of group samples is met, the detection efficiency is extremely low, the workload is large, time and labor are wasted, and the practicability is poor. Therefore, it is an urgent need to solve the technical problem of researchers in the field to provide a new molecular marker combination and a detection method thereof, so as to improve the sensitivity, accuracy and working efficiency, and to realize high-throughput typing detection and molecular marker-assisted screening of large-population samples.
The current common detection method for SNP is allele specific PCR (A1le adaptive PCR), and AS-PCR distinguishes SNP sites based on whether the 3' end base of a primer is matched with a DNA template or not. Theoretically, as long as the last base is not paired, the primer cannot be extended efficiently; however, the final single base mismatch often fails to achieve the desired discrimination. With exponential DNA amplification, the number of mismatched products is also considerable, to the extent that resolution is affected. In view of the above, the present invention is based on the correction mechanism of Pfu high fidelity DNA (deoxyribose nucleic acid) polymerase for mismatched bases at the 3 'end of the primer, and then the 3' end of the primer is modified by phosphorothioation, the paired modified primer obtains a product, and the unpaired modified primer is terminated by the polymerization reaction and thus there is no product. The dualization effect that the paired primers are extended and the unpaired primers are not extended perfectly meets the requirement of genotyping detection, and the false positive in a gene detection system is obviously reduced.
disclosure of Invention
the invention aims to provide a group of molecular marker primer combinations for identifying peach sticky nuclear separation traits and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that: a molecular marker primer combination for identifying peach sticky nuclear separation traits comprises a primer pair, wherein the primer pair is selected from one of the following three primer pairs:
JYSNP-1：F：5’-TGTAAAACGACGGCCAGTAGAGCCAATAATTTTA*G*G-3’，
R：5’-CAGAGTAAATGTTGTTAACCACT*T*G-3’；
JYSNP-2：F：5’-TGTAAAACGACGGCCAGTCAAAGATCGAGCCTTG*C*T-3’，
R：5’-CAATGCTTTTTTGGCTAAGCTAC*G*A-3’；
JYSNP-3：F：5’-TGTAAAACGACGGCCAGTCAAAGATCGAGCCTTG*C*T-3’，
R：5’-CAGAGTAAATGTTGTTAACCACT*T*G-3’；
wherein, is indicated as a thio modification.
preferably, the kit further comprises a tail primer, wherein the tail primer sequence is as follows:
Tail: 5 '- < FAM >/< HEX >/< NED >/< PET > TGTAAAACGACGGCCAGT-3', wherein 1-4 fluorescent labels can be selected optionally during synthesis;
The invention also discloses application of the molecular marker primer combination in identifying the peach sticky off-core character.
preferably, the steps include:
(1) Designing and synthesizing a primer: synthesizing one of the primers JYSNP-1, JYSNP-2, and JYSNP-3 of claim 1;
(2) Extraction of DNA: extracting genome DNA in the young peach leaves, wherein the young peach leaves refer to young peach leaves with the growth date of less than 20 days on the current branches;
(3) and (3) PCR amplification: performing PCR amplification by using the primer JYSNP-1, JYSNP-2 or JYSNP-3 synthesized in the step (1) based on the peach genome DNA;
(4) Detection and analysis of amplification products: and (4) carrying out agarose electrophoresis detection strip size analysis on the amplified product to determine the peach fruit nuclear sticking character.
the following steps may also be taken:
(1) Designing and synthesizing a primer: synthesizing one of the primers JYSNP-1, JYSNP-2, and JYSNP-3 of claim 1, and a tail primer;
(2) extraction of DNA: extracting genome DNA from the tender peach leaves;
(3) and (3) PCR amplification: performing PCR amplification by using the primer JYSNP-1, JYSNP-2 or JYSNP-3 synthesized in the step (1) based on the peach genome DNA;
(4) detection and analysis of amplification products: and analyzing the amplified product on an ABI genetic analyzer by using a tail primer to determine the peach fruit nuclear sticking character.
Preferably, the detailed steps are as follows:
(1) Primer synthesis: entrusting biotechnology companies to synthesize any one of primers JYSNP-1, JYSNP-2 and JYSNP-3 respectively; it should be noted that if the detection and analysis of the size of the PCR amplification product fragment is performed later by using a genetic analyzer, a tail primer needs to be synthesized separately, and one of four different fluorescent groups can be added to the 5' end of the tail primer, namely, one of FAM, HEX, PET and NED is selected;
(2) Extraction of DNA:
A. grinding young leaves of about 0.2g into powder by using liquid nitrogen, and placing the powder into a 2mL centrifuge tube;
B. adding 0.8mL of CTAB lysate preheated at 65 ℃ and uniformly mixing, wherein the formula of the lysate is as follows: 100mmol/L Tris. Cl (pH 8.0), 20mmol/L EDTA (pH 8.0), 1.4mmol/L NaCl, 2.5% CTAB (M/V), 3% PVP (M/V), 2% beta-mercaptoethanol (V/V); water bath at 65 deg.C for 1h, while shaking gently from time to time;
C. after the water bath is finished and the mixture is cooled, 0.8mL of extraction mixed solution is added and mixed uniformly, and the formula of the extraction mixed solution is as follows: chloroform: isoamyl alcohol 24: 1 (V/V); 4 ℃, 10000rpm, 10min, carefully transferring the supernatant into a new 2mL centrifuge tube, repeatedly adding 0.8mL extraction mixed solution, and uniformly mixing, 4 ℃, 10000rpm, 10 min;
D. carefully transferring the supernatant into a new 2mL centrifuge tube, adding isopropanol with the same volume (precooling at-20 ℃), mixing uniformly, and standing at room temperature for 30 min; carefully sucking out the flocculent precipitate, and washing with 70% ethanol;
E. after the residual ethanol is dried by an ultraclean workbench, 0.2mL of ddH2O is used for dissolving DNA; meanwhile, 1-2 mul of the DNA is taken to be detected on 1.0 percent agarose gel, and the DNA stock solution is diluted into working solution with the concentration of 80 ng/mul and stored in a refrigerator at the temperature of-20 ℃;
(3) The PCR amplification reaction system is as follows: genomic DNA template 1.0. mu.L (80 ng/. mu.L), 2 × Easypfu PCR Supermix 10. mu.L, forward primer (10. mu.M) 1. mu.L, reverse primer (10. mu.M) 1. mu.L, ddH2O to 20. mu.L; the PCR amplification reaction system is only suitable for detecting the size of a PCR product strip by conventional agarose electrophoresis; when the size of PCR product band is analyzed by ABI genetic analyzer, 0.5. mu.L of tail primer (10. mu.M) is added into the system, and the amount of forward primer is changed to 0.5. mu.L (10. mu.M). The amplification PCR procedure was as follows: 5min at 94 ℃, 30s at 52 ℃, 30s at 72 ℃, 35 cycles, 10min at 72 ℃;
(4) And (3) detecting and analyzing PCR amplification products: taking 5 mu L of PCR product obtained by the amplification process, and carrying out 1.2% agarose gel electrophoresis to detect the amplification effect; or detecting on an ABI genetic analyzer, and reading the fragment size of the data result by using software Genemapper or Peak Scanner; wherein the sizes of the fragments amplified by the JYSNP-1 primer, the JYSNP-2 primer and the JYSNP-3 primer are 178bp, 194bp and 106bp respectively. If any one of the corresponding primer bands obtained by PCR amplification is positive, the peach fruit is represented as a nuclear-abscission character, otherwise, the peach fruit is represented as a nuclear-adhesion character;
compared with the prior art, the invention has the beneficial effects that:
(1) the molecular marker primers JYSNP-1, JYSNP-2 and JYSNP-3 amplification products have stable and clear band types, good repeatability and easy resolution; in addition, the size difference of the product fragments after the primer amplification is obvious, and the detection is carried out by agarose electrophoresis; the primers can be independently used for molecular marker-assisted screening and can also be simultaneously used, so that the test cost is reduced, and the working efficiency is greatly improved;
(2) the molecular marker primers JYSNP-1, JYSNP-2 and JYSNP-3 in the invention have the amplification product fragment size of 100bp-200bp, are suitable for high-throughput typing detection technology platforms such as fluorescent capillary electrophoresis and fluorescent PCR instruments, further improve the genotyping accuracy and detection efficiency, and have reliable detection effect; meanwhile, a universal primer M13 sequence is added at the 5' end of the primers JYSNP-1, JYSNP-2 and JYSNP-3, and a Tail primer sequence (Tail) added with a fluorescent group is added for PCR amplification, so that the cost for synthesizing the fluorescent primers can be further reduced, only 1-4 Tail primers added with fluorescent marks need to be synthesized, and the primers JYSNP-1, JYSNP-2 and JYSNP-3 can be combined and used with specific molecular marker primers JYSNP-1, JYSNP-2 and JYSNP-3 randomly, so that the test cost is reduced;
(3) the invention is based on a correction mechanism of utilizing Pfu high fidelity DNA polymerase to mismatch bases at the 3 'end of the primer, and then carrying out double-thio phosphorylation modification on the 3' ends of the forward primer and the reverse primer, the matched modified primer obtains a product, and the unpaired modified primer is terminated with polymerization reaction, so that no product exists, the false positive in a gene detection system is obviously reduced, and the accuracy of genotyping detection is further improved.
The invention is described in more detail below with reference to the following examples and the accompanying drawings. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
drawings
FIG. 1 shows the agarose electrophoresis typing effect of PCR amplification products of JYSNP-2 and JYSNP-3 primers of peach germplasm resource DNA samples;
FIG. 2 shows the fluorescent capillary electrophoresis typing effect of PCR amplification products of JYSNP-1, JYSNP-2 and JYSNP-3 primers of peach germplasm resource DNA samples;
FIG. 3 shows the typing effect of JYSNP-2 primer of peach germplasm resource DNA sample on the platform of the fluorescence quantitative PCR instrument.
Detailed Description
Example 1
an application method of a group of molecular marker primer combinations for identifying peach sticky off-core traits comprises the following detailed steps:
(1) Primer synthesis: according to the sequence information in Table 1, primers JYSNP-1, JYSNP-2, JYSNP-3 and tail primers were synthesized by Compton Biotech engineering (Shanghai) Ltd, respectively; wherein, three different fluorescent groups, namely FAM, HEX and NED, are added at the 5' end of the tail primer;
table 1: related primer sequences
Note: indicated as thio modification.
(2) extracting DNA of peach leaves of a population sample to be detected:
A. grinding young leaves of about 0.2g into powder by using liquid nitrogen, and placing the powder into a 2mL centrifuge tube;
B. adding 0.8mL of CTAB lysate preheated at 65 ℃ and uniformly mixing, wherein the formula of the lysate is as follows: 100mmol/L Tris. Cl (pH 8.0), 20mmol/L EDTA (pH 8.0), 1.4mmol/L NaCl, 2.5% CTAB (M/V), 3% PVP (M/V), 2% beta-mercaptoethanol (V/V); water bath at 65 deg.C for 1h, while shaking gently from time to time;
C. after the water bath is finished and the mixture is cooled, 0.8mL of extraction mixed solution is added and mixed uniformly, and the formula of the extraction mixed solution is as follows: chloroform: isoamyl alcohol 24: 1 (V/V); 4 ℃, 10000rpm, 10min, carefully transferring the supernatant into a new 2mL centrifuge tube, repeatedly adding 0.8mL extraction mixed solution, and uniformly mixing, 4 ℃, 10000rpm, 10 min;
D. Carefully transferring the supernatant into a new 2mL centrifuge tube, adding isopropanol with the same volume (precooling at-20 ℃), mixing uniformly, and standing at room temperature for 30 min; carefully sucking out the flocculent precipitate, and washing with 70% ethanol;
E. after the residual ethanol is dried by an ultraclean workbench, 0.2mL of ddH2O is used for dissolving DNA; meanwhile, 1-2 mul of the DNA is taken to be detected on 1.0 percent agarose gel, and the DNA stock solution is diluted into working solution with the concentration of 80 ng/mul and stored in a refrigerator at the temperature of-20 ℃;
(3) The PCR amplification reaction system is as follows: genomic DNA template 1.0. mu.L (80 ng/. mu.L), 2 × Easypfu PCR Supermix 10. mu.L, forward primer (10. mu.M) 0.5. mu.L, tail primer (10. mu.M) 0.5. mu.L, reverse primer (10. mu.M)
1 μ L, ddH2O to 20 μ L; the amplification PCR procedure was as follows: 5min at 94 ℃, 30s at 52 ℃, 30s at 72 ℃, 35 cycles, 10min at 72 ℃;
(4) and (3) detecting and analyzing PCR amplification products:
the PCR product is detected on an ABI 3730 genetic analyzer (ABI company, USA), and can be subjected to PCR amplification by using tail primers added with three different fluorophores, or tail primers added with the same fluorophore, so that the test cost is reduced; mixing products obtained by PCR amplification by using different specific primers, detecting on an ABI genetic analyzer, and reading the size of the fragments by using software Genemapper or Peak Scanner according to data results; wherein the sizes of the fragments amplified by the JYSNP-1 primer, the JYSNP-2 primer and the JYSNP-3 primer are 178bp, 194bp and 106bp respectively. If any one of the primer bands corresponding to the size is obtained by PCR amplification, the result is positive (peach fruit is a nuclear separation property), otherwise, the result is a nuclear adhesion property, two reference samples (NJCT1 and NJCT2) are added for each batch of typing identification, namely, the property is determined to be a nuclear separation detection sample, if the reference samples are amplified to be positive, the result is acceptable, otherwise, the test is failed, and corresponding test data cannot be used; finally, the results of the examination of the sticky nuclear character of all the test specimens are recorded in table 2, and the specific typing effect is shown in fig. 2.
TABLE 2 information of the identification results of the stuck nuclear of 70 peach germplasm resources used in the examples of the present invention
note: the presence and absence of amplification products of expected fragment size are indicated by "+" and "-" respectively.
Example 2
An application method of a group of molecular marker primer combinations for identifying peach sticky off-core traits comprises the following detailed steps:
(1) primer synthesis: according to the sequence information in Table 1, the related biotechnology company is requested to synthesize any one of the primers JYSNP-1, JYSNP-2, and JYSNP-3 (in this example, only the primer JYSNP-2 is synthesized);
Table 1: related primer sequences
Note: indicated as thio modification.
(2) Extracting DNA of peach leaves of a population sample to be detected:
A. grinding young leaves of about 0.2g into powder by using liquid nitrogen, and placing the powder into a 2mL centrifuge tube;
B. adding 0.8mL of CTAB lysate preheated at 65 ℃ and uniformly mixing, wherein the formula of the lysate is as follows: 100mmol/L Tris. Cl (pH 8.0), 20mmol/L EDTA (pH 8.0), 1.4mmol/L NaCl, 2.5% CTAB (M/V), 3% PVP (M/V), 2% beta-mercaptoethanol (V/V); water bath at 65 deg.C for 1h, while shaking gently from time to time;
C. After the water bath is finished and the mixture is cooled, 0.8mL of extraction mixed solution is added and mixed uniformly, and the formula of the extraction mixed solution is as follows: chloroform: isoamyl alcohol 24: 1 (V/V); 4 ℃, 10000rpm, 10min, carefully transferring the supernatant into a new 2mL centrifuge tube, repeatedly adding 0.8mL extraction mixed solution, and uniformly mixing, 4 ℃, 10000rpm, 10 min;
D. Carefully transferring the supernatant into a new 2mL centrifuge tube, adding isopropanol with the same volume (precooling at-20 ℃), mixing uniformly, and standing at room temperature for 30 min; carefully sucking out the flocculent precipitate, and washing with 70% ethanol;
E. after the residual ethanol is dried by an ultraclean workbench, 0.2mL of ddH2O is used for dissolving DNA; meanwhile, 1-2 mul of the DNA is taken to be detected on 1.0 percent agarose gel, and the DNA stock solution is diluted into working solution with the concentration of 80 ng/mul and stored in a refrigerator at the temperature of-20 ℃;
(3) the PCR amplification reaction system is as follows: genomic DNA template 1.0. mu.L (80 ng/. mu.L), 2 XPuper EvaGreen Master Mix for HRM PCR Super Mix 5. mu.L, forward primer (10. mu.M) 0.5. mu.L, reverse primer (10. mu.M)
0.5. mu.L, 10 × ROX reference dye ddH2O to 10. mu.L; based on an ABI Q6Flex real-time fluorescent quantitative PCR system platform, the Genotyping experiment type 'Genotyping' is used; the amplification PCR procedure was as follows: 5min at 94 ℃, 30s at 52 ℃, 30s at 72 ℃, 40 cycles, and finally 10min at 72 ℃, and collecting fluorescence signals;
(4) genotyping analysis and identification results: taking 5 mu L of PCR product obtained by the amplification process, and carrying out 1.2% agarose gel electrophoresis to detect the amplification effect; if the method is adopted to detect the sizes of the product fragments, two pairs of specific primers, namely JYSNP-2 and JYSNP-3, can be directly used, and can be directly distinguished on agarose electrophoresis due to the large size difference of the two fragments, and meanwhile, PCR amplification products of the two primers can be detected by electrophoresis by using the same lane, so that the detection is not mutually influenced, and the labor and the time are saved; if any one of the corresponding primer bands is obtained by PCR amplification, the positive result is obtained (the peach fruit is in the nuclear-abscission property), otherwise, the sticky-core property is obtained; it is noted that two reference samples (NJCT1 and NJCT2) are required to be added for each batch of typing identification, namely a detection sample with the characteristic determined as nuclear separation is obtained, if the amplification of the reference sample is positive, the test result is acceptable, otherwise, the test fails, and the corresponding test data cannot be used; finally, the results of the identification of the sticky nuclear character of all the other test samples under the condition that the reference sample is amplified to be positive are recorded by using a table, and the specific resolution effect is shown in figure 1;
continuing to carry out typing identification analysis under the condition that the analysis identification result of the reference sample is credible; the method is characterized in that the sample adherence nucleus character can be known based on Allelic diagnosis Plot in the automatic analysis result of an ABI Q6Flex real-time fluorescent quantitative PCR system platform (ABI company in America); wherein, the groups divided in the same region with the reference samples (NJCT1 and NJCT2) are the nuclear separation characters, the rest are the sticky nuclei (see figure 3), and the final result is recorded in a table form, see table 2.
TABLE 2 information on the identification results of the stuck nuclear of 76 peach germplasm resources used in the examples of the present invention
note: the presence and absence of amplification products of expected fragment size are indicated by "+" and "-" respectively.
Claims (8)
1. a molecular marker primer combination for identifying peach sticky nuclear separation traits comprises a primer pair, wherein the primer pair is selected from one of the following three primer pairs:
JYSNP-1：F：5’-TGTAAAACGACGGCCAGTAGAGCCAATAATTTTA*G*G-3’，
R：5’-CAGAGTAAATGTTGTTAACCACT*T*G-3’；
JYSNP-2：F：5’-TGTAAAACGACGGCCAGTCAAAGATCGAGCCTTG*C*T-3’，
R：5’-CAATGCTTTTTTGGCTAAGCTAC*G*A-3’；
JYSNP-3：F：5’-TGTAAAACGACGGCCAGTCAAAGATCGAGCCTTG*C*T-3’，
R：5’-CAGAGTAAATGTTGTTAACCACT*T*G-3’；
wherein, is indicated as a thio modification.
2. the molecularly imprinted primer combination of claim 1, wherein: the kit also comprises a tail primer, wherein the sequence of the tail primer is as follows:
Tail：5’-<FAM>/<HEX>/<NED>/<PET>TGTAAAACGACGGCCAGT-3’。
3. Use of the molecular marker primer combination of claim 1 or 2 for identifying the peach stick-off nucleus trait.
4. the use according to claim 3, comprising the steps of:
(1) designing and synthesizing a primer: synthesizing one of the primers JYSNP-1, JYSNP-2, and JYSNP-3 of claim 1;
(2) extraction of DNA: extracting genome DNA from the tender peach leaves;
(3) and (3) PCR amplification: performing PCR amplification by using the primer JYSNP-1, JYSNP-2 or JYSNP-3 synthesized in the step (1) based on the peach genome DNA;
(4) Detection and analysis of amplification products: and (4) carrying out agarose electrophoresis detection strip size analysis on the amplified product to determine the peach fruit nuclear sticking character.
5. The use according to claim 3, comprising the steps of:
(1) Designing and synthesizing a primer: synthesizing one of the primers JYSNP-1, JYSNP-2, and JYSNP-3 of claim 1, and a tail primer;
(2) Extraction of DNA: extracting genome DNA from the tender peach leaves;
(3) And (3) PCR amplification: performing PCR amplification by using the primer JYSNP-1, JYSNP-2 or JYSNP-3 synthesized in the step (1) based on the peach genome DNA;
(4) Detection and analysis of amplification products: and analyzing the amplified product on an ABI genetic analyzer by using a tail primer to determine the peach fruit nuclear sticking character.
6. Use according to claim 4 or 5, characterized in that: the extraction of the DNA in the step (2) is specifically as follows:
A. grinding about 0.2g of tender leaves by using liquid nitrogen into powder, and placing the powder into a 2mL centrifuge tube;
B. adding 0.8mL of CTAB lysate preheated at 65 ℃ and uniformly mixing, wherein the formula of the lysate is as follows: 100mmol/L Tris. Cl, pH 8.0, 20mmol/L EDTA, pH 8.0, 1.4mmol/L NaCl, 2.5% CTAB (M/V), 3% PVP (M/V), 2% beta-mercaptoethanol (V/V); water bath at 65 deg.C for 1h, while shaking gently from time to time;
C. after the water bath is finished and the mixture is cooled, 0.8mL of extraction mixed solution is added and mixed uniformly, and the formula of the extraction mixed solution is as follows: chloroform: isoamyl alcohol 24: 1 (V/V); centrifuging at 4 deg.C and 10000rpm for 10min, transferring the supernatant into a new 2mL centrifuge tube, repeatedly adding 0.8mL extraction mixture, mixing, and centrifuging at 4 deg.C and 10000rpm for 10 min;
D. Transferring the supernatant into a new 2mL centrifuge tube, adding equal volume of isopropanol precooled at-20 ℃, mixing uniformly, and standing at room temperature for 30 min; sucking out the flocculent precipitate, and washing with 70% ethanol;
E. after the residual ethanol is dried by an ultraclean workbench, 0.2mL of ddH2O is used for dissolving DNA; meanwhile, 1-2 mul of the DNA is taken to be detected on 1.0 percent agarose gel, and the DNA stock solution is diluted into working solution with the concentration of 80 ng/mul and stored in a refrigerator at the temperature of-20 ℃;
7. Use according to claim 4, characterized in that: the reaction system of PCR amplification in the step (3) is as follows: 80 ng/. mu.L of genomic DNA template 1.0. mu.L, 2 × Easypfu PCR Supermix 10. mu.L, 10. mu.M forward primer 1. mu.L, 10. mu.M reverse primer 1. mu.L, ddH2O to 20. mu.L; the amplification PCR procedure was as follows: 94 ℃ for 5min, 94 ℃ for 30s, 52 ℃ for 30s, 72 ℃ for 30s, 35 cycles, 72 ℃ for 10 min.
8. use according to claim 5, characterized in that: the reaction system of PCR amplification in the step (3) is as follows: 80 ng/. mu.L of genomic DNA template 1.0. mu.L, 2 × Easypfu PCR Supermix 10. mu.L, 10. mu.M forward primer 0.5. mu.L, 10. mu.M reverse primer 1. mu.L, 10. mu.M tail primer 0.5. mu.L, ddH2O to 20. mu.L; the amplification PCR procedure was as follows: 94 ℃ for 5min, 94 ℃ for 30s, 52 ℃ for 30s, 72 ℃ for 30s, 35 cycles, 72 ℃ for 10 min.
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