CN113736912B - Primer group for constructing molecular marker map of tobacco variety after baking Hunan main-cultivated tobacco variety and application of primer group - Google Patents
Primer group for constructing molecular marker map of tobacco variety after baking Hunan main-cultivated tobacco variety and application of primer group Download PDFInfo
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Abstract
The invention discloses a primer pair for constructing a molecular marker map of a tobacco leaf variety after the tobacco variety is baked in Hunan province, the sequence of the primer pair is shown as SEQ ID No.1-46, and the primer pair can be used for identifying Hunan tobacco No. 3, hunan tobacco No. 5, hunan tobacco No. 6, hunan tobacco No. 7, yunnan tobacco No. 87, yunnan tobacco No. 97, yunnan tobacco No. 99, yunnan tobacco No. 100, yunnan tobacco No. 203, zhongyan tobacco No. 100, guangdong tobacco No. 9, guiyan tobacco No.1, safflower Dajinyuan, longjiang 911, nanjiang No. 3, NC729, G80 or K326 of the tobacco variety after the tobacco variety is baked in Hunan province. Has important significance for helping tobacco enterprises to rapidly, accurately and efficiently identify and classify cured tobacco leaves.
Description
Technical Field
The invention relates to the technical field of tobacco variety identification, in particular to a primer group for constructing a molecular marker map of a tobacco variety after curing of a Hunan main-cultivated tobacco variety and application thereof.
Background
Tobacco leaves are the basis for the development of the tobacco industry, and the grade quality of the tobacco leaves is the life line of the tobacco industry. The quality condition and the industrial availability of the tobacco grades are finally reflected on the demands and the utilization values of tobacco raw materials in the cigarette industry, the cigarette quality and the brand development are directly influenced, and the health, stability and sustainable development of the whole tobacco industry are influenced. Tobacco classification is affected by a variety of factors, such as production factors and artifacts. Production factors, such as natural factors, ecological factors, environmental conditions, etc., have a relatively obvious effect on tobacco production, but many of these are uncontrollable factors. Human factors such as variety selection, cultivation management, tobacco baking, identification and grading and the like are not only core bases for improving tobacco quality, but also important factors for influencing the tobacco quality.
The variety represents the hereditary property and the seed property of tobacco leaves, is a relatively stable gene foundation and is also a key for influencing the identification and classification of tobacco leaves. The tobacco variety has different plant height, leaf number, growth period, baking characteristic and the like, and the produced leaves have different sizes, widths and thicknesses, so that the quality of tobacco leaves is different, the authenticity of the tobacco variety is the basis of stable leaf group formula, and the product quality and the style are ensured. The traditional purchasing grading is carried out by means of eye observation and hand touch, and the quality cannot be measured by means of human qualitative recognition. Tobacco grading becomes more complex, increasing the difficulty and uncertainty of grading and grading. The modern molecular marking technology can accurately and effectively distinguish the variety authenticity of tobacco, and provides reliable evidence for tobacco identification and classification.
The basis of molecular markers is the acquisition of DNA. Under natural conditions, DNA (deoxyribonucleic acid) of the plant leaves after being isolated is slowly degraded under the action of nuclease, and the baking process accelerates the degradation rate of the tobacco leaf DNA to a certain extent and has great influence on subsequent PCR (polymerase chain reaction), so that the extraction of the tobacco leaf DNA after baking is a main limitation of limiting the application of molecular markers on the identification of the tobacco leaves after baking. The method used to extract genomic DNA is different depending on the object of investigation, purpose of investigation, cost of investigation, etc. of the plant. Currently, plant genomic DNA extraction methods are numerous, such as cetyltrimethylammonium bromide (CTAB) method, sodium Dodecyl Sulfate (SDS) method, polyvinylpyrrolidone (PVP) method. The different methods have advantages and disadvantages, wherein the CTAB method has the best effect and the most wide application. The method is used for fresh plants, and DNA extraction is simpler. After baking, the external morphology and internal chemical composition of tobacco leaves are greatly changed, and especially DNA is greatly degraded. In addition, the sugar and phenol substances in the cured tobacco have obvious interference effect on DNA extraction, so that the cured tobacco is more difficult to extract than the DNA of fresh tobacco. Guo Zhaokui and the like extract roasted tobacco DNA by 8 methods of WJC method, oard and Dronova1, gibco plant DNA extraction kit, edward, qiagen D Neasy plant mini kit, modified CTAB method, SDS method and modified phenol/simulated extraction method, determine the concentration and purity thereof, analyze and evaluate the quality of the extracted DNA by combining the chloroplast non-coding protein sequence and the PCR amplification result of 35S promoter sequence, and find that the amplified band obtained by the modified CTAB method and QIAGEN DNeasyPlantMiniKit extracted DNA is optimal. Sun Jiu and the like, the main strips of the genome DNA of the primary cured tobacco leaves extracted by the magnetic bead method are clear, no obvious impurity strips exist, and the purity is high. The roasted tobacco leaf DNA extracted by the column type plant genome DNA extraction kit such as Xuanbeibei has higher purity, and the adopted commercial kit can better remove protein, phenols and other substances in the tobacco leaf, has better removal effect on residual ethanol, chloroform and the like in the extraction process, and the extracted DNA can meet the subsequent test requirements of molecular marker screening and the like. Although many methods are capable of extracting DNA meeting the PCR requirements, all methods require liquid nitrogen for milling, and are limited in the purchase of inconvenient units of liquid nitrogen. Almost all methods require about 50mg of extraction material, greatly limiting the amount of extracted DNA, and are not applicable to cases where there is a large need for multiple primer screening and templates. Besides, the price of the kit is high, the 50 times of extraction kits are generally between 400 and 500 yuan, the magnetic bead method is also required to extract the tobacco, the requirements are high, and the cost investment for tobacco enterprises with large identification amount can be greatly increased.
Molecular markers are specific DNA fragments that reflect a certain difference in the genome of an individual or population of an organism. The varieties are distinguished by detecting the sizes and the numbers of the different fragments among the different varieties, which is similar to the fingerprints of human beings, so that the polymorphism of the DNA fragments of the different varieties is also referred to as the fingerprint. Depending on the means and type of the generation of the differential fragments, the molecular markers include AFLP, RFLP, SCAR, SSR and SNP, etc. SSR (simple sequence repeat) molecular markers have the advantages of high polymorphism, codominance inheritance, simple and rapid technology, good repeatability, abundant quantity and the like, and have been widely applied to tobacco fingerprint construction and variety distinction. Liu Guoxiang and the like utilize 25 pairs of SSR primers to carry out genetic diversity analysis on 33 sun-cured tobacco varieties, and successfully construct fingerprint spectrums of the 33 sun-cured tobacco varieties; xu Jun and the like screen 8 pairs of SSR primers with better polymorphism, and completely distinguish 80 tobacco germplasm; yin Guoying et al screen 14 pairs of core primers from 278 pairs of SSR primers for tobacco germplasm resource identification, and so on. In summary, the SSR molecular marking technology can effectively distinguish different tobacco varieties, and is simple to operate. Notably, the above studies have been conducted primarily using fresh tobacco leaves as the material, but the tobacco industry enterprises use cured tobacco leaves. The research on whether the cured tobacco DNA can be continuously used for molecular marking and the like is relatively less how much the tobacco DNA is degraded in the curing process. The Xuanbeibei and the like analyze the genetic diversity of 9 flue-cured tobacco varieties by utilizing SSR markers. And screening to effectively distinguish 8 pairs of SSR primers for 9 flue-cured tobacco varieties. Sun Jiu and the like take 11 primary tobacco varieties commonly used by cigarette enterprises as materials, and 5 pairs of primer pairs with high polymorphism and strong stability are screened from 24 pairs of candidate SSR primer pairs. On the other hand, reported researches mainly focus on identification of few cultivars or germplasm resources, and identification researches on a plurality of main cultivars are lacking, so that the significance of classification guidance on tobacco leaf identification of a flue-cured tobacco enterprise is not strong. In conclusion, a set of detection system based on an SSR molecular marker technology is established for the Hunan main cultivated tobacco variety. Has important significance for helping tobacco enterprises to rapidly, accurately and efficiently identify and classify cured tobacco leaves.
Disclosure of Invention
Aiming at the defect that tobacco leaf identification and grading of a flue-cured tobacco enterprise lacks an efficient grading detection method, the invention establishes a detection system based on an SSR molecular marker technology aiming at the main cultivated tobacco variety in Hunan. Has important significance for helping tobacco enterprises to rapidly, accurately and efficiently identify and classify cured tobacco leaves.
The SSR primer for constructing the molecular marker map of the tobacco variety after the tobacco variety is baked in Hunan main cultivation is as follows:
| number | Name | Forward primer | Reverse primer |
| P2 | PT30159 | GCATGCATATGAACATGGGA | TTTGACATCTCTACTCTTCCGTTT |
| P3 | PT30339 | AGAGTTTGGTCCTTTAATGCG | AAAGTTCCTGTTCAATAGCGATG |
| P4 | PT30424 | TTTGGAATCAATAAGACGACAA | TTGACCAGTAGGCTTATCACACA |
| P5 | PT20021 | TCAAAGTCTTTTACATTGACG | GGCAATGTTGTGGCTTAC |
| P6 | PT20382 | TTCACAGGGTGGGAAAATGT | ACTCCTAAACCTCGCCCAAC |
| P7 | PT30230 | TTTCTTTCTGTCTGATGCTTCAAT | TTGTCCATCTCACTTGCTGC |
| P8 | PT30399 | TGTGTGCACCCTCCAATTTA | TGATCTCTAGAGTGGTGGCATC |
| P9 | PT20189 | AAAGGTTCGGTATCCCAG | ATTGGACGATGAGAACGA |
| P10 | PT30028 | AAACTTGAAGCAGAGACGGC | GCACATGCGGATCTTGATTT |
| P11 | PT30292 | AAGACAGATTGGTGCGGAAC | AGCACTTGGACAGGCGAATA |
| P12 | PT30421 | CGTACCCTGAATGCCATCTT | ATGCAGCGTTTCAGGAATTT |
| P13 | PT1037 | CAAATATCCAACACCCCA | CGGAGTGAAAGTGGTGAG |
| P14 | PT20306 | CCGAGTCTGTTTTGGTTG | GCGAGCATCTCTCATTTC |
| P15 | PT30224 | CTTTGCAGCAACAATCTCAA | CACTTGGCTAGGCTAAATAAGCA |
| P16 | PT30380 | AACGAGTGTAAACATGCTCCC | CCACCAGCACAATAATGAACA |
| P17 | PT20172 | ACACCTCCTTCTTCCTGC | CCAAAATGGTTCACTGGA |
| P18 | PT30021 | CATTTGAACATGGTTGGCTG | CTCAACTCTCGTCGCTCTTG |
| P19 | PT30259 | CAGCCAAGAGAACCCTTCAG | GATTACCCTTCAAATGCCGA |
| P20 | PT30403 | CCAACTCTACCGCTAACTTCAAA | CACGACTGACGAGACATGGT |
| P21 | PT40024 | AATGTCTGCCCAATCGAAAG | CGAATAACGACACTCGAACG |
| P22 | PT30470 | TTTAAGCTCATTTGAGCCCG | CGTATTTGAGATTTATATGCCTTCG |
| P23 | PT20165 | TGTCTCGTGAAGCATGAA | GGAAATGGAGGATCTCGT |
| P24 | PT40021 | TCAAATCAAATCAACCCTCTCC | TGGTTGGAGCTTCTCTCGTT |
These primer pairs can be used in tobacco variety identification or in molecular map building blocks after curing of Hunan main cultivated tobacco variety. The Hunan main cultivated tobacco varieties are Hunan tobacco No. 3, hunan tobacco No. 5, hunan tobacco No. 6, hunan tobacco No. 7, cloud tobacco 87, cloud tobacco 97, cloud tobacco 99, cloud tobacco 100, cloud tobacco 203, medium tobacco 100, guangdong tobacco 9, guiyan tobacco No.1, safflower Dajinyuan, longjiang 911, nan Jiang No. 3, NC729, G80 or K326.
The construction method of the molecular marker map of the tobacco leaf variety after the tobacco variety is baked in Hunan main planting comprises the following steps:
(1) Extracting DNA of the cured tobacco leaves;
(2) Taking the extracted DNA as a template, taking a primer group as an amplification primer, establishing a PCR reaction system and carrying out PCR amplification;
(3) And (3) carrying out electrophoresis on the amplified products, and forming primer letter numbers and band type numbers of amplified band position numbers on the amplified result of the sample DNA of each variety according to the electrophoresis result.
Preferably, the DNA of the cured tobacco leaves is extracted by adopting a CTAB method;
the PCR amplification system (10. Mu.L) was 2X M5 Taq PCR Mix 10. Mu. L, P1 1.3. Mu. L, P2 2.0.3. Mu. L, DNA 2. Mu.L (20 ng/ul), dd H 2 O, 7.2. Mu.L; the amplification procedure was: 95. at the temperature of 3 min; 95. 30℃, s;53 ℃,30 s;72 ℃,30s,35 cycles; 72℃for 10 min.
A kit for identifying the tobacco variety after baking of Hunan main-cultivated tobacco variety comprises the primer pair.
The beneficial effects are that:
the invention establishes a detection system based on an SSR molecular marker technology aiming at the main cultivated tobacco variety in Hunan. Has important significance for helping tobacco enterprises to rapidly, accurately and efficiently identify and classify cured tobacco leaves.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 shows gel detection after SDS and kit extraction of DNA and RNase treatment;
FIG. 2 shows a flocculent DNA obtained by adding isopropanol during the extraction of DNA by CTAB method;
FIG. 3 shows the extraction of DNA from different sites by CTAB method;
FIG. 4 shows the detection of DNA extraction effect using SSR primers;
FIG. 5 shows the amplification effect of primers at different annealing temperatures;
FIG. 6 shows amplification maps of different primers of the same variety;
FIG. 7 shows polymorphism analysis of 5 pairs of main primer pairs for test varieties;
FIG. 8 shows polymorphism analysis of 5 pairs of auxiliary primers;
FIG. 9 is a map of a part of a polymorphism-free primer;
fig. 10 is a flow chart of test tobacco identification.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and explanation only and is not intended to limit the present invention.
Examples
Cultivation of tobacco
Sterilizing tobacco seeds with 20% NaClO for 20min, washing with sterile water for 3-4 times, re-suspending with 0.2% agar, spreading on solid 1/2MS culture medium (2.4 g MS salt, 3% sucrose, 0.8% agar powder, pH 5.8,121 deg.C, sterilizing for 15-20 min), sealing with sealing film, and placing in incubator. The culture conditions are: culturing at 25deg.C for 12 hr in darkness and 12 hr in light for 15-20 days, and planting in basin containing nutrient soil (nutrient soil: vermiculite=1:1). After 10 days, it can be used for DNA extraction.
Extraction of DNA
(1) Weighing 0.1. 0.1 g-0.3. 0.3 g tobacco, cutting with scissors, placing in a mortar, adding appropriate liquid nitrogen, grinding into powder, rapidly transferring into 2 mL centrifuge tube, adding 600 μL CTAB extract preheated to 65deg.C and containing 1% beta-mercaptoethanol and 6 μL mg ·mL -1 After shaking vigorously using a vortex shaker for 1 minute, treating in a 65 ℃ water bath for 10 minutes; (2) 200. Mu.L of a mixed solution of chloroform and isoamyl alcohol (24:1) was added, and the mixture was vortexed for 1 minute, followed by 12000 rpm at 4℃for 15 minutes; (3) mu.L of the supernatant was aspiratedAdding 200 mu L of isopropanol precooled at 4 ℃ into a 1.5 mL centrifuge tube, and properly shaking up and down; (4) Centrifuging at 4deg.C and 12000 rpm for 15 min, removing supernatant, retaining white flocculent precipitate at bottom, adding 70% ethanol precooled at 4deg.C, and rinsing white precipitate by shaking; (5) After repeating the step (4) once, the supernatant is removed, dried, and 100. Mu.L of sterilized double distilled water is added to dissolve the precipitate, and the precipitate is preserved at-20 ℃.
Detecting the quality of the genomic DNA by using 1% agarose gel electrophoresis; the genomic DNA concentration of each material was measured by a spectrophotometer and diluted to 20 ng. Mu.l -1 As an amplified template for SSR.
PCR and electrophoresis
PCR reaction System for primer amplification (10. Mu.L)
| Composition of the components | Volume (mu l) | Concentration of mother liquor |
| PCR mix | 5 | 10× |
| P F | 0.3 | 10 µM |
| P R | 0.3 | 10 µM |
| H 2 O | 2.4 | |
| DNA | 2 | 20ng·ul -1 |
PCR amplification was performed on Eppendorf T100, with the following amplification procedure:
electrophoresis: preparation of 4% agarose gel: 160ml of electrophoresis buffer (1 xTAE) was measured and placed in a 250ml conical flask, 6.4g of agarose was weighed and heated to complete dissolution by a microwave oven (note: benefits after gel boiling prevention, timely opening the microwave oven when a large number of bubbles emerge after boiling, stopping heating, and continuing heating after the bubbles fall). Adding 10-15 mu l EB, uniformly mixing, and rapidly pouring into a glue making groove with two rows of comb holes. After the gel is completely solidified (the gel is milk white) the comb is pulled out for electrophoresis. 100 Electrophoresis is carried out for 1.5-2 hours under the voltage of V until bromophenol blue runs to the bottom layer of the gel, and the electrophoresis is stopped. And observing the electrophoresis strip by using a gel imaging system, photographing and storing.
SSR primers used in the study were as follows:
| number | Name | Forward primer | Reverse primer |
| P2 | PT30159 | GCATGCATATGAACATGGGA | TTTGACATCTCTACTCTTCCGTTT |
| P3 | PT30339 | AGAGTTTGGTCCTTTAATGCG | AAAGTTCCTGTTCAATAGCGATG |
| P4 | PT30424 | TTTGGAATCAATAAGACGACAA | TTGACCAGTAGGCTTATCACACA |
| P5 | PT20021 | TCAAAGTCTTTTACATTGACG | GGCAATGTTGTGGCTTAC |
| P6 | PT20382 | TTCACAGGGTGGGAAAATGT | ACTCCTAAACCTCGCCCAAC |
| P7 | PT30230 | TTTCTTTCTGTCTGATGCTTCAAT | TTGTCCATCTCACTTGCTGC |
| P8 | PT30399 | TGTGTGCACCCTCCAATTTA | TGATCTCTAGAGTGGTGGCATC |
| P9 | PT20189 | AAAGGTTCGGTATCCCAG | ATTGGACGATGAGAACGA |
| P10 | PT30028 | AAACTTGAAGCAGAGACGGC | GCACATGCGGATCTTGATTT |
| P11 | PT30292 | AAGACAGATTGGTGCGGAAC | AGCACTTGGACAGGCGAATA |
| P12 | PT30421 | CGTACCCTGAATGCCATCTT | ATGCAGCGTTTCAGGAATTT |
| P13 | PT1037 | CAAATATCCAACACCCCA | CGGAGTGAAAGTGGTGAG |
| P14 | PT20306 | CCGAGTCTGTTTTGGTTG | GCGAGCATCTCTCATTTC |
| P15 | PT30224 | CTTTGCAGCAACAATCTCAA | CACTTGGCTAGGCTAAATAAGCA |
| P16 | PT30380 | AACGAGTGTAAACATGCTCCC | CCACCAGCACAATAATGAACA |
| P17 | PT20172 | ACACCTCCTTCTTCCTGC | CCAAAATGGTTCACTGGA |
| P18 | PT30021 | CATTTGAACATGGTTGGCTG | CTCAACTCTCGTCGCTCTTG |
| P19 | PT30259 | CAGCCAAGAGAACCCTTCAG | GATTACCCTTCAAATGCCGA |
| P20 | PT30403 | CCAACTCTACCGCTAACTTCAAA | CACGACTGACGAGACATGGT |
| P21 | PT40024 | AATGTCTGCCCAATCGAAAG | CGAATAACGACACTCGAACG |
| P22 | PT30470 | TTTAAGCTCATTTGAGCCCG | CGTATTTGAGATTTATATGCCTTCG |
| P23 | PT20165 | TGTCTCGTGAAGCATGAA | GGAAATGGAGGATCTCGT |
| P24 | PT40021 | TCAAATCAAATCAACCCTCTCC | TGGTTGGAGCTTCTCTCGTT |
2. Experimental results
1. Method for extracting DNA of cured tobacco leaves
1. Fumbling for extracting DNA of cured tobacco leaves by different methods
The DNA of the cured tobacco leaves is extracted by adopting three general methods of SDS, CTAB and kit, and the difference of extraction effects is large. All three methods can extract DNA, and DNA bands are in a diffuse form, and the diffuse bands cannot be removed by RNase treatment and chloroform extraction, which indicates that the DNA is degraded in a large amount. The DNA extraction method of the kit is the most simple and convenient, but the extraction material cannot exceed 20mg, the extraction amount is small, the main band and the dispersion band are not obvious, and the kit is about 50 times more expensive. The SDS method has simple operation method, more impurities, especially polysaccharide, and the extract is transparent and viscous and is not easy to suck. During the operation, a large amount of bubbles are generated by adding chloroform for extraction, and the subsequent operation is influenced. Electrophoresis of the extract showed significant dispersion bands, but the main bands were not. The modified CTAB development was somewhat complicated, but could handle 1g of material. The extract shows strong bands after electrophoresis, and the main band is obvious.
2. Fumbling for extracting DNA of different types of materials by CTAB method
And taking upper and middle leaves and tobacco leaf powder of the upper, middle, lower and green cigarettes of the normal cigarettes. During the pyrolysis precipitation process, the 6 types of materials can all generate flocculent precipitation after isopropanol is added (figure 2). The electrophoresis results show that different types of materials can possibly extract DNA, but the extraction effects are relatively different. 1, but the concentration of the 2 extracted degraded DNA with low molecular weight is highest, and the main band part is also bright. 3 and 5, but the total DNA amount is less. The 4 bands are very bright, but the main band portion is not clear. 6 has the worst effect, the main band is not obvious, and the total DNA amount is small. 1-6, indicating that the extracted DNA also had impurities that were not likely to be too much of the treated sample, too much impurities, and not completely removed (fig. 3). Although the grinding workload is reduced after the components are ground by the grinder, the grinder is not recommended because the grinder cannot thoroughly clean the components after grinding and can pollute the results. On the other hand, the extraction effect of the powder (6) is also the worst, probably because the sample after pulverization accelerates degradation of DNA during storage.
3. PCR detection of DNA extracted by CTAB method
Three pairs of SSR primers are synthesized, and PCR is carried out by taking DNA extracted by a CTAB method as a template, and 3% agarose gel electrophoresis detection is carried out. The results showed clear bands with weak dispersion in individual lanes, but without affecting the observations. From the image it can be seen that PT 201665 can divide 6 materials into two groups, 1, 4, 6 having one band, 2, 3, 5 having two bands, proving that the material taken does not originate from 1 variety. PT20172 has one band and PT20213 has one band, but two pairs of primers have the same band type among six materials and have no polymorphism (FIG. 4). The result shows that DNA extracted by the CTAB method can meet the PCR requirement, and the variety of the cured tobacco leaves can be distinguished by the SSR primer.
2. Extraction of fresh leaf DNA of test variety
The DNA of fresh tobacco leaves is extracted by adopting a CTAB method, and the quality and the concentration of the DNA are detected by utilizing agarose gel electrophoresis and a spectrophotometer. The result shows that the DNA concentration difference is larger, the DNA concentration of Xiang tobacco No. 6 is lowest and is 139.97, the original DNA concentration of Zhongyan tobacco 100 is the largest and is 1291.07, but the DNA quality is better and the tailing degradation phenomenon is avoided. Taking part of the DNA stock solution, diluting to 20 ng/. Mu.l, using for PCR detection, and storing the rest in a refrigerator at-80 ℃.
The concentration of DNA extracted from 19 varieties of fresh tobacco leaves is as follows:
| variety of species | Concentration (ng/. Mu.l) | Numbering device | Variety of species | Concentration (ng/. Mu.l) |
| Hunan tobacco No. 3 | 488.28 | 11 | Zhongyan 100 | 1144.64 |
| Hunan cigarette No. 5 | 696.92 | 12 | Yue cigarette 97 | 918.12 |
| Hunan tobacco No. 6 | 139.97 | 13 | Noble cigarette number one | 854.43 |
| Hunan tobacco No. 7 | 557.81 | 14 | Safflower Dajinyuan | 177.77 |
| Cloud cigarette 87 | 467.97 | 15 | Longjiang 981 | 480.32 |
| Cloud cigarette 97 | 458.66 | 16 | Nanjiang No. 3 | 616.39 |
| Cloud cigarette 99 | 290.1 | 17 | NC297 | 1030.05 |
| Cloud cigarette 100 | 588.74 | 18 | G80 | 619.05 |
| Cloud tobacco 203 | 416.67 | 19 | K326 | 452.78 |
| Zhongyan 100 Yuan (Chinese traditional medicine) | 1291.07 |
3. Primer pair fire temperature detection
Primer 23 pairs were synthesized for the first time and the primer annealing temperature was detected. The amplification effect of different primers is greatly different at the annealing temperature of 50-56 ℃. Some primers are unable to amplify bands such as 2, 6, 23 at 50 ℃; most primers do not amplify bands at 54 and 56 ℃; at 53 ℃; most primers can amplify the band, and the main band is clear. The annealing temperature was set to 53℃and a round of verification was performed.
4. Primary screening of primers
The 19 materials are used for screening 23 pairs of primers, so that most primers have no polymorphism among varieties or are not obvious, and few primers have better polymorphism, which indicates that the relatedness of the tobaccos is relatively close. Through screening, 1, 2, 3, 7, 8, 21, 22, 23 are selected for further verification. And (3) PCR amplification: amplification system (10 μl): 2X M5 Taq PCR Mix 10 mu L, P1 0.3 mu L, P2.3 mu L, DNA mu L (20 ng/ul), dd H 2 O, 7.2. Mu.L. The amplification procedure was: 95. at the temperature of 3 min; 95. 30℃, s;53 ℃,30 s;72 ℃,30s,35 cycles; 72℃for 10 min. Electrophoresis conditions: preparing 4% agarose gel: 6.4g agarose was weighed out and dissolved in 160mL 1xTAE buffer. The solution was heated in a microwave oven to no water (observed with gentle shaking during heating), 14 μl of EB solution was rapidly added, the agarose solution was poured into the electrophoresis tank, a small-hole comb was inserted, and the gel was cooled until it was completely coagulated. 10. Mu.L of the PCR product was put into the gel well (10. Mu.L of DNA marker was put into the gel well). 100V, electrophoresis for 2h, then using gelAnd (5) observing by a glue imaging system and photographing.
Second round of primer verification
We used 19 test materials to perform polymorphism analysis on the candidate primers to distinguish the test materials. 1. 2, 6, 7, 9, 11, 14, 22, 23 have polymorphism, and primers such as 3, 8, 17, 19 have no polymorphism among different varieties. 17 varieties can be separated by using 1, 9, 10, 21, 23 main primers. Performing auxiliary identification on some varieties by using 5 pairs of auxiliary primers, wherein the auxiliary primers are required to be added for determining 2 and 19; 14 and 17 are assisted by P7 and P22; 6 and 9 require P7 assistance and 7 require P2 assistance. The specific classification process comprises the following steps: (1) with primer 23, all varieties can be classified into three categories: the first class is 1, 2, 3, 5, 6, 7, 9, 14, 15, 17, 18, 19, the second class is 4, 8, 10, 11, 13, 16, and the third class is only 12; (2) using primer 1, 2, 15, 18, 19 in the first class can be identified, the remaining 1, 3, 5, 6, 7, 9 cannot be separated, and 14 and 17 cannot be separated. It can be identified that 4, 8, 10, 13, 11 and 16 in the second class cannot be separated. (3) Using primer 21, 3 of the first class can be identified, the remaining 1, 5, 7 being one class, 6 and 9 being one class, 14 and 17 being one class, indistinguishable. 11, 16 in the second class can be identified. (4) With primer 9, 1, 5, 7 can be identified, while distinguishing 6 from 9, 14 and 17 into one class, and not distinguishing. (5) With primer 10, 14 and 17 can be distinguished.
The fingerprint patterns of the 19 tobacco varieties are as follows:
| variety of species | Atlas | Variety of species | Atlas |
| 1 | A23BM1234C13D123E1 | 11 | A13BM123C12D124E2 |
| 2 | A23BU3C3D124E2 | 12 | A123BM123C14D124E2 |
| 3 | A13BM1C34D123E2 | 13 | A13BU3C0D123E1 |
| 4 | A23BU4C3D124E1 | 14 | A23BU2C2D124E2 |
| 5 | A23BM123C13D124E2 | 15 | A23BM1C13D123E2 |
| 6 | A23BM123C3D124E1 | 16 | A13BM124C14D124E1 |
| 7 | A23BM123C13D124E1 | 17 | A23BU3C2D124E1 |
| 8 | A13BM123C13D123E2 | 18 | A23BU1C2D124E1 |
| 9 | A23BM1C12D123E1 | 19 | A23BM124C12D123E2 |
| 10 | A13BU3C2C2D124E2 |
Annotation: p23 (A), P1 (B), P21 (C), P9 (D), P10 (E), M (Multiple) represents a plurality of bands, U (Unique) represents a single band; 1-4 represent the sequence numbers of the stripes, from large to small.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Hunan tobacco Co Yongzhou Co Ltd
Xiao Qinzhi
<120> primer set for constructing molecular marker map of tobacco leaf variety after curing Hunan main-cultivated tobacco variety and application thereof
<160> 46
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gcatgcatat gaacatggga 20
<210> 2
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tttgacatct ctactcttcc gttt 24
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
agagtttggt cctttaatgc g 21
<210> 4
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
aaagttcctg ttcaatagcg atg 23
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
tttggaatca ataagacgac aa 22
<210> 6
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
ttgaccagta ggcttatcac aca 23
<210> 7
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
tcaaagtctt ttacattgac g 21
<210> 8
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
ggcaatgttg tggcttac 18
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
ttcacagggt gggaaaatgt 20
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
actcctaaac ctcgcccaac 20
<210> 11
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
tttctttctg tctgatgctt caat 24
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
ttgtccatct cacttgctgc 20
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
tgtgtgcacc ctccaattta 20
<210> 14
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
tgatctctag agtggtggca tc 22
<210> 15
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
aaaggttcgg tatcccag 18
<210> 16
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
attggacgat gagaacga 18
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
aaacttgaag cagagacggc 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
gcacatgcgg atcttgattt 20
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
aagacagatt ggtgcggaac 20
<210> 20
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
agcacttgga caggcgaata 20
<210> 21
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
cgtaccctga atgccatctt 20
<210> 22
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
atgcagcgtt tcaggaattt 20
<210> 23
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
caaatatcca acacccca 18
<210> 24
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
cggagtgaaa gtggtgag 18
<210> 25
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
ccgagtctgt tttggttg 18
<210> 26
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
gcgagcatct ctcatttc 18
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
ctttgcagca acaatctcaa 20
<210> 28
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
cacttggcta ggctaaataa gca 23
<210> 29
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
aacgagtgta aacatgctcc c 21
<210> 30
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
ccaccagcac aataatgaac a 21
<210> 31
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
acacctcctt cttcctgc 18
<210> 32
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
ccaaaatggt tcactgga 18
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
catttgaaca tggttggctg 20
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
ctcaactctc gtcgctcttg 20
<210> 35
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
cagccaagag aacccttcag 20
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
gattaccctt caaatgccga 20
<210> 37
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
ccaactctac cgctaacttc aaa 23
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
cacgactgac gagacatggt 20
<210> 39
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
aatgtctgcc caatcgaaag 20
<210> 40
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
cgaataacga cactcgaacg 20
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
tttaagctca tttgagcccg 20
<210> 42
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
cgtatttgag atttatatgc cttcg 25
<210> 43
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
tgtctcgtga agcatgaa 18
<210> 44
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
ggaaatggag gatctcgt 18
<210> 45
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
tcaaatcaaa tcaaccctct cc 22
<210> 46
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
tggttggagc ttctctcgtt 20
Claims (4)
1. Constructing a primer pair of a molecular marker map of a tobacco leaf variety after baking a Hunan main-cultivated tobacco variety, which is characterized by comprising 5 pairs of primers contained in SEQ ID No.1 and SEQ ID No.2, SEQ ID No.17 and SEQ ID No.18, SEQ ID No.19 and SEQ ID No.20, SEQ ID No.41 and SEQ ID No.42, and SEQ ID No.45 and SEQ ID No. 46;
the Hunan main cultivated tobacco varieties are Hunan tobacco No. 3, hunan tobacco No. 5, hunan tobacco No. 6, hunan tobacco No. 7, cloud tobacco 87, cloud tobacco 97, cloud tobacco 99, cloud tobacco 100, cloud tobacco 203, medium tobacco 100, guangdong tobacco 9, guiyan tobacco No.1, safflower Dajinyuan, longjiang 911, nan Jiang No. 3, NC729, G80 or K326.
2. Use of the primer pair according to claim 1 in tobacco variety identification after tobacco variety baking in Hunan province, wherein the Hunan province tobacco variety is Hunan province tobacco variety No. 3, hunan province tobacco No. 5, hunan province tobacco No. 6, hunan province tobacco No. 7, cloud tobacco 87, cloud tobacco 97, cloud tobacco 99, cloud tobacco 100, cloud tobacco 203, medium tobacco 100, guangdong tobacco 9, guiyan tobacco No.1, safflower Dajinyuan, longjiang 911, nanjiang No. 3, NC729, G80 or K326.
3. The use of the primer pair according to claim 1 in constructing molecular patterns of tobacco varieties after baking of Hunan main-cultivated tobacco varieties, wherein the Hunan main-cultivated tobacco varieties are Hunan tobacco No. 3, hunan tobacco No. 5, hunan tobacco No. 6, hunan tobacco No. 7, yunnan tobacco No. 87, yunnan tobacco No. 97, yunnan tobacco No. 99, yunnan tobacco No. 100, yunnan tobacco No. 203, zhongyan tobacco No. 100, yue tobacco No. 9, guiyan No.1, honghuadajinyuan, longjiang 911, nan Jiang No. 3, NC729, G80 or K326.
4. A kit for identifying a cured tobacco variety of a major cured tobacco variety of a hunan comprising the primer pair of claim 1.
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