Response surface method-based flue-cured tobacco leaf molecular identification method and application

Abstract

The invention discloses a response surface method-based flue-cured tobacco leaf molecular identification method and application. The method comprises the following steps: mixing a to-be-detected sample with a DNA extracting solution, heating in a water bath, analyzing conditions for extracting DNA of the to-be-detected sample by a single factor, optimizing the extraction conditions by a response surface method, determining an optimal extraction method according to a regression equation, and identifying a molecular marker. According to the method, the influence of the sample adding amount, the water bath time and the centrifugal time on the concentration and the quality of the DNA is analyzed through a single-factor experiment, on the basis, a Box-Behnken response surface method is used for optimizing the DNA extraction method of the flue-cured tobacco leaves, the optimal conditions for extracting the DNA of the flue-cured tobacco leaves are determined, the concentration of the extracted DNA is high, operation is easy, and cost is low.

Description

Technical Field

[0001] The invention belongs to the technical field of DNA extraction and relates to a molecular identification method for flue-cured tobacco leaves based on a response surface methodology and an application thereof. Background Art

[0002] The quality of tobacco leaves reflects the comprehensive indicators of tobacco leaves and affects the quality and brand development of cigarette products. In recent years, the raw materials of the cigarette industry have shown unstable quality and unclear variety identification. It is difficult to grade and judge by traditional visual and hand touch methods. How to efficiently and accurately identify the raw materials of tobacco leaves purchased after baking is a difficult problem that industrial companies urgently need to solve.

[0003] Molecular marker technology has been widely used in the construction of fingerprints and genetic diversity analysis of flue-cured tobacco, cigars, sun-dried tobacco, aromatic tobacco, wild tobacco, etc. However, most of the above studies use fresh leaves as materials. The appearance of flue-cured tobacco leaves changes significantly, and DNA will also be severely degraded, which has a great impact on subsequent PCR and other experiments. At present, the main methods for DNA extraction from flue-cured tobacco leaf samples include CTAB method, SDS method, magnetic bead method and kit. Compared with the CTAB method, the SLS method takes less time and is more efficient to extract DNA. It does not require the addition of chemical reagents such as β-mercaptoethanol that are harmful to the human body, and is cheaper than the kit and magnetic bead method. However, the quality of the extracted DNA from flue-cured tobacco leaves is low, which is difficult to meet the molecular identification of flue-cured tobacco leaves.

[0004] The Box-Behnken design response surface methodology is a second-order experimental design method based on three levels. It has the advantages of a small number of experiments and high experimental precision. It is often used in the research of active substance extraction process optimization, chemical synthesis and purification process in the fields of pharmacy, chemistry, food, etc.

[0005] Therefore, there is an urgent need to provide a method for DNA extraction and identification of flue-cured tobacco leaves using the Box-Behnken response surface model. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides a molecular identification method for flue-cured tobacco leaves based on response surface methodology and its application, determines the optimal conditions for extracting DNA from flue-cured tobacco leaves, and the extracted DNA has high concentration, simple operation and low cost.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for identifying flue-cured tobacco leaves, the method comprising: mixing a sample to be tested with a DNA extracting solution, heating in a water bath, analyzing the conditions for extracting DNA from the sample to be tested by single factor analysis, optimizing the extraction conditions by response surface methodology, determining the best extraction method based on a regression equation, and molecular marker identification.

[0009] The invention analyzes the influence of sample addition amount, water bath time and centrifugation time on DNA concentration and quality through single factor experiment, and optimizes the DNA extraction method of flue-cured tobacco leaves by using Box-Behnken response surface method on this basis, and determines the optimal conditions for extracting DNA from flue-cured tobacco leaves, and the extracted DNA has high concentration, simple operation and low cost.

[0010] Preferably, the DNA extraction solution contains EDTA, Tris-HCl, NaCl and SLS.

[0011] Preferably, the final concentration of EDTA in the DNA extraction solution is 0.01-0.05M (eg, 0.01M, 0.02M, 0.03M, 0.04M, 0.05M).

[0012] Preferably, the concentration of Tris-HCl in the DNA extract is 0.05-0.2M (eg, 0.05M, 0.06M, 0.08M, 0.1M, 0.15M, 0.2M).

[0013] Preferably, the concentration of NaCl in the DNA extract is 0.05-0.2M (eg, 0.05M, 0.06M, 0.08M, 0.1M, 0.15M, 0.2M).

[0014] Preferably, the concentration of SLS in the DNA extraction solution is 5-15 g / L (e.g., 5 g / L, 6 g / L, 8 g / L, 10 g / L, 12 g / L, 15 g / L).

[0015] Preferably, the conditions for extracting DNA from the sample to be tested by single factor analysis include: 260 / A 280 As the evaluation index, a single factor test was carried out with sample volume, water bath time and centrifugation time as independent variable factors.

[0016] Preferably, the added amount is 20-40 mg (eg, 20 mg, 30 mg, 40 mg).

[0017] Preferably, the water bath time is 10-20 min (eg 10 min, 15 min, 20 min).

[0018] Preferably, the centrifugation time is 8-12 min (eg 8 min, 10 min, 12 min).

[0019] In the present invention, the DNA concentration and A 260 / A 280As the evaluation index, a single-factor experiment was carried out with the sample amount (10 mg, 20 mg, 40 mg, 60 mg, 80 mg), water bath time (0 min, 10 min, 20 min, 30 min), and centrifugation time (6 min, 8 min, 10 min, 12 min) as factors to determine the effect of each factor on DNA concentration.

[0020] Preferably, the response surface method for optimizing the extraction conditions comprises: based on the single factor experiment, optimizing the extraction conditions based on the DNA concentration and A 260 / A 280 The response value was taken as the factor, and a 3-factor 3-level Box-Behnken test was carried out with sample volume, water bath time and centrifugation time as the factors. The optimal extraction conditions were determined according to the regression equation.

[0021] Preferably, the regression equation includes Y1=694.9+134.86A-15.91B-11.55C-29.96AB-36.56AC+25.32BC-300.02A 2 -133.84B 2 -203.69C 2 (Formula 1) and Y2 = 1.72 - 0.1650A - 0.0136B + 0.0046C + 0.0012AB - 0.0249AC + 0.0550BC - 0.0793A 2 -0.0830B 2 -0.1280C 2 (Formula 2);

[0022] Among them, Y1 represents DNA concentration, Y2 represents A 260 / A 280 A represents the sampling volume, B represents the centrifugation time, and C represents the water bath time.

[0023] Preferably, the method for determining the optimal extraction conditions is to use DNA concentration and A 260 / A 280 The maximum value of the ratio is used to determine the sampling volume, centrifugation time and water bath time.

[0024] In the present invention, the optimal extraction conditions are as follows: the sample volume is 20.894 mg, the centrifugation time is 10.412 min, the water bath time is 16.433 min, the theoretical concentration of DNA extraction and A 260 / A 280 The ratios were 601.005 ng / μL and 1.728, respectively. To facilitate practical operation, the extraction conditions were simplified to 20 mg sample volume, 10 min centrifugation time, and 15 min water bath time. Five parallel tests were performed according to this method, and the DNA concentration obtained was 588.16 ± 27.09 ng / μL, A260 / A 280 It is 1.75±0.03, which has a small error with the concentration estimated by the software, indicating that the model has a high degree of fit with the actual experimental results and the method is reliable.

[0025] Preferably, the molecular marker identification comprises performing a KASP-PCR reaction to detect SNP typing.

[0026] Preferably, the reaction procedure of the KASP-PCR reaction includes: denaturation at 94-96°C (e.g., 94°C, 95°C, 96°C) for 10-15 min (e.g., 10 min, 12 min, 15 min); denaturation at 94-96°C (e.g., 94°C, 95°C, 96°C) for 10-20 s (e.g., 10 s, 15 s, 20 s), annealing at 60-63°C (e.g., 60°C, 61°C, 63°C) for 50-60 s (e.g., 50 s, 55 s, 60 s), for a total of 10-15 cycles (e.g., 10 cycles, 12 cycles, 15 cycles), starting from the 2nd cycle, the annealing temperature decreases by 0.5-0.6°C each cycle (e.g. 0.5°C, 0.55°C, 0.6°C); denaturation at 94-96°C (e.g. 94°C, 95°C, 96°C) for 15-20s (e.g. 15s, 18s, 20s), annealing at 53-55°C (e.g. 53°C, 54°C, 55°C) for 50-60s (e.g. 50s, 55s, 60s), for a total of 24-26 cycles (e.g. 24 cycles, 25 cycles, 26 cycles).

[0027] Preferably, the flue-cured tobacco leaves include any one of Yunyan 87, Honghua Dajinyuan, CB-1, Qinyan 96 or Zhongyan Texiang 301, or a combination of at least two of them.

[0028] In a second aspect, the present invention provides application of the method described in the first aspect in identification of flue-cured tobacco leaf varieties.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention analyzes the effects of sample addition amount, water bath time, and centrifugation time on DNA concentration and quality through single factor experiments, and on this basis, optimizes the DNA extraction method of flue-cured tobacco leaves using the Box-Behnken response surface methodology, and determines the optimal conditions for extracting DNA from flue-cured tobacco leaves. The extracted DNA has high concentration, simple operation, and low cost;

[0031] (2) The present invention provides a mature, reliable and efficient technical method for molecular identification of flue-cured tobacco leaf raw materials, which can realize the tracking of the entire process from field management to acquisition and sampling, provide technical support for the molecular identification of flue-cured tobacco leaf varieties, and provide guarantee for the stability and reliability of raw material quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a curve chart showing the effect of different sample addition amounts on the DNA concentration and quality of flue-cured tobacco leaves;

[0033] Figure 2 This is a curve diagram showing the effect of different water bath time on the DNA concentration and quality of flue-cured tobacco leaves;

[0034] Figure 3 This is a curve diagram showing the effect of different centrifugation times on the DNA concentration and quality of flue-cured tobacco leaves;

[0035] Figure 4 The response surface diagram of the interaction between sample volume and centrifugation time on DNA extraction concentration;

[0036] Figure 5 The response surface diagram of the interaction between sample volume and water bath time on DNA extraction concentration;

[0037] Figure 6 This is the response surface diagram of the interaction between water bath time and centrifugation time on DNA extraction concentration;

[0038] Figure 7 A is the effect of sample volume and centrifugation time 260 / A 280 The interaction response surface plot of ;

[0039] Figure 8 A is the effect of sample volume and water bath time 260 / A 280 The interaction response surface plot of ;

[0040] Fig. 9 The effect of water bath time and centrifugation time on A 260 / A 280 The interaction response surface plot of ;

[0041] Fig.10 This is the capillary electrophoresis detection diagram of the amplification results using DNA extracted by SLS method as template;

[0042] Fig.11 The capillary electrophoresis detection diagram is a result of amplification using the DNA extracted by the method of the present invention as a template;

[0043] Fig. 12A This is the genotyping diagram of the results of the KASP marker CigarSNP03-1 testing DNA extracted by the SLS method;

[0044] Fig. 12B This is the genotyping diagram of the results of the KASP marker CigarSNP06-2 testing DNA extracted by the SLS method;

[0045] Fig. 12CThis is the genotyping diagram of the KASP marker CigarSNP11-1 test results of DNA extracted by SLS method;

[0046] Fig.12D This is a genotyping diagram of the detection results of the DNA extracted by the method of the present invention using the KASP marker CigarSNP03-1;

[0047] Fig.12E The genotyping diagram of the detection result of the DNA extracted by the method of the present invention using the KASP marker CigarSNP06-2;

[0048] Fig.12F The genotyping diagram of the detection result of KASP marker CigarSNP11-1 on DNA extracted by the method of the present invention;

[0049] Fig.13 This is a schematic gel diagram of the capillary electrophoresis detection of the PT20192 primer amplification results;

[0050] Fig.14 This is a clustering diagram of 15 tobacco leaf samples based on SSR markers. DETAILED DESCRIPTION

[0051] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0052] Example 1

[0053] The test materials in this example are flue-cured tobacco leaf samples of Yunyan 87, K326 and Zhongyan 100, which are sourced from the Southwest Experimental Base of the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences.

[0054] (1) DNA extraction and quality testing

[0055] The invention optimizes key steps on the basis of a conventional sodium lauryl sulfate (SLS) extraction method. The conventional SLS extraction method is as follows: take the sample and place it in a 2mL centrifuge tube, load it with steel beads, cool it with liquid nitrogen, and grind it with a sampler (Shanghai Jingxin Industrial Development Co., Ltd., Tissuelyser-48); add 800μL of SLS extraction solution (100mL 0.2M EDTA+100mL 1MTris-HCl+100mL 1M NaCl+10g SLS, make up to 1000mL), and shake it for 2min; add 800μL of phenol-chloroform-isoamyl alcohol (V / V / V=25:24:1), and shake it for 2min; centrifuge it at 12000rpm for 10min; aspirate 600μL of supernatant into a new 1.5mL centrifuge tube, add an equal volume of cold isopropanol (-20℃) to precipitate DNA; centrifuge it at 12000rpm for 10min, discard the supernatant, wash it once with 75% ethanol, dry it, and add 100μL ddH2O to dissolve it.

[0056] Genomic DNA integrity test. 5 μL sample + 1 μL 6× Loading buffer was spotted into the wells of 1% agarose gel, and electrophoresis was performed at 150V for 15 min using 1×TAE buffer. After EB staining, the gel was imaged on a gel imager (Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd., JY04S-3E). The marker was GL5000 DNAMarker (Acray Bio, AG11906).

[0057] Genomic DNA concentration and purity detection. Use UV spectrophotometer to measure the concentration of genomic DNA and record A 230 , A 260 and A 280 , calculate A 260 / A 280 and A 260 / A 230 SPSS Statistics 28.0.0.0 software was used for variance analysis.

[0058] (2) Single factor test

[0059] Single factor experiments were designed to optimize the extraction conditions from three aspects: sample addition, water bath time, and centrifugation time. Sample addition optimization: The sample addition of the flue-cured tobacco leaves was 10 mg, 20 mg, 40 mg, 60 mg, and 80 mg respectively; after adding the SLS extract, a water bath lysis step was added, 60°C water bath for 0 min, 10 min, 20 min, and 30 min; centrifugation at 12000 rpm for 6 min, 8 min, 10 min, and 12 min.

[0060] (3) Response surface experiment

[0061] Taking the results of the single factor experiment as a reference, DNA concentration (Y1) and A 260 / A 280 (Y2) is the response value, and the three influencing factors of sample volume (A), centrifugation time (B), and water bath time (C) are independent variables. The Box-Behnken experimental design principle in the software Design Expert 11 is used to set the levels of the factors (Table 1).

[0062] Table 1

[0063]

[0064] The test was carried out according to the 17 test points obtained by Design Expert 11 software. The scheme and results are shown in Table 2.

[0065] Table 2

[0066]

[0067]

[0068] The experimental results were analyzed using Design Expert 11 software (Table 3), and the DNA concentration (Y1) and A 260 / A 280 The three-variable quadratic regression equation of (Y2) and sampling volume (A), centrifugation time (B), and water bath time (C) is: Y1 = 694.9 + 134.86A-15.91B-11.55C-29.96AB-36.56AC+25.32BC-300.02A 2 -133.84B 2 -203.69C 2 , Y2=1.72-0.1650A-0.0136B+0.0046C+0.0012AB-0.0249AC+0.0550BC-0.0793A 2 -0.0830B 2 -0.1280C 2 The p values ​​of the two models were both less than 0.0001, indicating that they were significant. The p values ​​of the lack of fit terms were 0.6415 and 0.1569, respectively, both greater than 0.05, indicating that the random error of the experiment had no significant effect on the optimization test results. The DNA concentration model R 2 (R-Squared, coefficient of determination) = 0.9813, R adj 2 (Adjusted R-Squared, Corrected Determination Coefficient) = 0.9572, A 260 / A 280 Model R 2=0.9777, R adj 2 =0.9490(R 2 ), are both close to 1, indicating that the fitting degree of the two models is good and can be used to predict the actual test results.

[0069] From the response surface diagram of the interaction of various factors, it can be seen that ( Figure 4-9 ), the response surface of AC interaction is the steepest and the contour lines are the most dense, while BC is the smoothest and the contour lines are the most dispersed, indicating that the order of significance of the interaction is AC>AB>BC. Therefore, the order of priority of the three factors is sampling volume>water bath time>centrifugation time.

[0070] Table 3

[0071]

[0072]

[0073] (4) Verification of optimal parameters of extraction method

[0074] DNA concentration and A 260 / A 280 The maximum value of the ratio was the target. The optimal parameters of the extraction method were obtained by Design Expert 11 software as follows: sampling volume 20.894 mg, centrifugation time 10.412 min, water bath time 16.433 min, the theoretical concentration of DNA extraction and A 260 / A 280 The ratios were 601.005 ng / μL and 1.728, respectively. To facilitate practical operation, the extraction conditions were simplified to 20 mg sample volume, 10 min centrifugation time, and 15 min water bath time. Five parallel tests were performed according to this method, and the DNA concentration obtained was 588.16 ± 27.09 ng / μL, A 260 / A 280 It is 1.75±0.03, which has a small error with the concentration estimated by the software, indicating that the model has a high degree of fit with the actual experimental results and the optimization method is reliable.

[0075] (5) SSR molecular marker detection

[0076] In order to detect whether the quality of DNA extracted from flue-cured tobacco leaves by the method of the present invention can meet the requirements of capillary electrophoresis detection, flue-cured tobacco leaves of Yunyan 87, K326 and Zhongyan 100 were used as materials, and DNA was extracted by the SLS method before and after optimization, respectively, and capillary electrophoresis detection was performed using primers PT51378 (SEQ ID NO.17 and SEQ ID NO.30).

[0077] The amplification system was 10 μL, including 1 μL DNA, 5 μL 2× RapidTaq Master Mix, 1 μL upstream primer, 1 μL downstream primer, and 2 μL ddH2O. The reaction procedure was: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s; 55°C annealing for 30 s; 72°C extension for 30 s, 35 cycles; 72°C extension for 8 min.

[0078] 2 μL of PCR product and 22 μL of 1×TE Buffer were mixed and added to the first 95 wells of a 96-well plate, and 24 μL of 35-400 bp DNA Ladder was added to the last well. The plate was then placed in a Fragment Analyzer fully automatic capillary electrophoresis instrument for detection.

[0079] PROSize 3.0 software was used to analyze SSR amplification data, and SSR capillary electrophoresis was used to detect amplified fragment peaks. The abscissa was the fragment size, and the ordinate was the fluorescence intensity value.

[0080] The results of capillary electrophoresis showed that the fluorescence value of the target fragment amplified using the DNA extracted by SLS method as a template was extremely low and the peak was not obvious ( Fig.10 ); and using the DNA extracted by the SLS method optimized by the method of the present invention as a template, a significant target fragment can be amplified ( Fig.11 ).

[0081] (6) SNP molecular marker detection

[0082] Flue-cured tobacco leaves of Yunyan 87, K326 and Zhongyan 100 were used as materials, with 6 replicates for each material. DNA was extracted using the SLS method and the method of the present invention, and SNP loci were detected using KASP primers.

[0083] The KASP-PCR reaction was carried out on a 384-well PCR instrument (Eppendorf Mastercycler pro), and the reaction system was 5 μL: 1 μL DNA (50 ng / μL) dried, 2.5 μL KASP Master mix (2×), 0.07 μL primer mixture (prepared by mixing Primer AlleteX, Primer AlleteY, and Primer Common at a concentration of 100 μmol / L in a volume ratio of 2:2:5, and the specific sequence is shown in Table 4), and the rest was filled with ddH2O.

[0084] Table 4

[0085]

[0086] The KASP-PCR amplification program was as follows: the first stage was denaturation at 94°C for 15 min; the second stage was denaturation at 94°C for 20 s and annealing at 61°C for 60 s, for a total of 10 cycles (starting from the second cycle, the annealing temperature decreased by 0.6°C each cycle); the third stage was denaturation at 94°C for 20 s and annealing at 55°C for 60 s, for a total of 26 cycles.

[0087] The KASP-PCR amplification products were read by fluorescence signal using a BMG POLARstar Omega scanner. The SNP typing results were analyzed using KlusterCaller 3.4.1 software (KBioscience): the genotypes clustered near the X-axis were the alleles connected to the FAM fluorescent tag sequence, and the genotypes clustered near the Y-axis were the alleles connected to the HEX fluorescent tag sequence.

[0088] The results of SNP marker detection showed that when DNA extracted by SLS method was used as template, there were many signal points drifting away in the genotyping graph, and the signal graph was scattered ( Figure 12A-12C ); The typing diagram obtained by using the DNA extracted by the method of the present invention as a template can clearly type the test materials ( Figure 12D-12F ).

[0089] Example 2

[0090] Five flue-cured tobacco varieties from different regions were used to test the practicality of DNA extraction. Each variety included one fresh tobacco leaf sample and two flue-cured tobacco leaf samples. The specific information is shown in Table 5.

[0091] Table 5

[0092]

[0093]

[0094] (1) DNA extraction

[0095] The DNA extraction procedure is the same as in Example 1.

[0096] (2) PCR amplification

[0097] The PCR reaction system is the same as in Example 1.

[0098] (3) SSR molecular marker detection

[0099] Using flue-cured tobacco leaf DNA from Yunyan 87, K326 and Zhongyan 100 as templates, 13 pairs of primers were screened by capillary electrophoresis (Table 6). The flue-cured tobacco leaf samples in Table 4 were tested by capillary electrophoresis. The data were analyzed using PRO Size 3.0 software to read the fragment size of the amplified product. PowerMarker V3.25 was used to draw the UPGMA cluster analysis diagram.

[0100] Table 6

[0101]

[0102]

[0103] The capillary electrophoresis results showed that ( Fig.13 ), the target bands can be amplified from both fresh and flue-cured tobacco leaves; cluster analysis results showed that ( Fig.14 ), the same flue-cured tobacco varieties from different sources are grouped into one category, which can provide a basis for the identification of flue-cured tobacco leaf varieties.

[0104] In summary, the present invention analyzes the effects of sample addition amount, water bath time and centrifugation time on DNA concentration and quality through single factor experiments, and on this basis uses the Box-Behnken response surface methodology to optimize the DNA extraction method of flue-cured tobacco leaves, and determines the optimal conditions for extracting DNA from flue-cured tobacco leaves. The extracted DNA has high concentration, simple operation and low cost.

[0105] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for identifying flue-cured tobacco leaves, characterized in that: The method comprises: mixing the sample to be tested with a DNA extracting solution, heating in a water bath, single factor analysis of the conditions for extracting DNA from the sample to be tested, optimizing the extraction conditions using a response surface method, determining the best extraction method according to a regression equation, and molecular marker identification.

2. The method according to claim 1, characterized in that The DNA extraction solution contains EDTA, Tris-HCl, NaCl and SLS.

3. The method according to claim 2, characterized in that The final concentration of EDTA in the DNA extraction solution is 0.01-0.05M; Preferably, the concentration of Tris-HCl in the DNA extraction solution is 0.05-0.2M; Preferably, the concentration of NaCl in the DNA extract is 0.05-0.2M; Preferably, the concentration of SLS in the DNA extraction solution is 5-15 g / L.

4. The method according to any one of claims 1 to 3, characterized in that The conditions for extracting DNA from the sample to be tested by single factor analysis include: DNA concentration and A 260 / A 280 As the evaluation index, a single factor test was conducted with the sample volume, water bath time, and centrifugation time as independent variable factors; Preferably, the sample addition amount is 20-40 mg; Preferably, the water bath time is 10-20 min; Preferably, the centrifugation time is 8-12 min.

5. The method according to any one of claims 1 to 4, characterized in that The response surface method for optimizing extraction conditions includes: based on single factor experiments, the DNA concentration and A 260 / A 280 As the response value, a 3-factor 3-level Box-Behnken test was performed with sample volume, water bath time, and centrifugation time as factors, and the optimal extraction conditions were determined according to the regression equation; Preferably, the regression equation includes Y1=694.9+134.86A-15.91B-11.55C-29.96AB-36.56AC+25.32BC-300.02A 2 -133.84B 2 -203.69C 2 (Formula 1) and Y2 = 1.72 - 0.1650A - 0.0136B + 0.0046C + 0.0012AB - 0.0249AC + 0.0550BC - 0.0793A 2 -0.0830B 2 -0.1280C 2 (Formula 2); Among them, Y1 represents DNA concentration, Y2 represents A 260 / A 280 A represents the sampling volume, B represents the centrifugation time, and C represents the water bath time.

6. The method according to claim 5, characterized in that The method for determining the optimal extraction conditions is to use DNA concentration and A 260 / A 280 The maximum value of the ratio is used to determine the sampling volume, centrifugation time and water bath time.

7. The method according to any one of claims 1 to 6, characterized in that The molecular marker identification includes performing KASP-PCR reaction to detect SNP typing.

8. The method according to any one of claims 1 to 7, characterized in that The reaction procedure of the KASP-PCR reaction includes: denaturation at 94-96°C for 10-15min; denaturation at 94-96°C for 10-20s, annealing at 60-63°C for 50-60s, for a total of 10-15 cycles, starting from the second cycle, the annealing temperature decreases by 0.5-0.6°C each cycle; denaturation at 94-96°C for 15-20s, annealing at 53-55°C for 50-60s, for a total of 24-26 cycles.

9. The method according to any one of claims 1 to 8, characterized in that The flue-cured tobacco leaves include any one of Yunyan 87, Honghua Dajinyuan, CB-1, Qinyan 96 or Zhongyan Texiang 301, or a combination of at least two of them.

10. Use of the method according to any one of claims 1 to 9 in identifying varieties of flue-cured tobacco leaves.

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