CN107354201B - Primer combination and kit for identifying flue-cured tobacco yunyan 97, application and detection method - Google Patents
Primer combination and kit for identifying flue-cured tobacco yunyan 97, application and detection method Download PDFInfo
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Abstract
The invention relates to a primer combination and a kit for identifying flue-cured tobacco yunyan 97, and an application and a detection method thereof, belonging to the technical field of tobacco variety identification. The method utilizes a tobacco 420K high-density SNP chip to carry out whole genome SNP typing on main tobacco cultivars in China in recent years, obtains a set of 13 specific SNP markers suitable for identifying flue-cured tobacco Yunyan 97 according to the inter-species polymorphism SNP site screening, and compares 13 SNP site flanking sequences obtained by cultivating a tobacco reference genome, wherein the SNP site flanking sequences are shown as SEQ ID NO. 1-13, and primers are designed based on the sequences; and carrying out typing detection on the sites by using the designed primers and adopting a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology. The SNP typing result is obtained through detection, and whether the sample is Yunyan 97 or not is identified.
Description
Technical Field
The invention relates to a primer combination and a kit for identifying flue-cured tobacco yunyan 97, and an application and a detection method thereof, belonging to the technical field of tobacco variety identification.
Background
Tobacco is an important economic crop and is a main raw material for cigarette production. The cured tobacco variety Yunyan 97 is bred by taking Yunyan 85 as a female parent and CV87 as a male parent by Yunnan tobacco agricultural science research institute through hybridization. Approved by the national tobacco variety approval committee at 3, 10 months in 2009. The plant is in a tower shape, the average topping plant height is 115.3cm, about 20 effective leaves are provided, the pitch is 5.7cm, and the stem circumference is 9.7 cm; the shape of the leaf is oblong, the color of the leaf is green, the leaf surface is slightly wrinkled, the leaf tip is blunt and sharp, the leaf edge is wavy, the main vein is medium in thickness, the leaf ear is medium, the waist leaf is 73.9cm long and the width is 30.3 cm; transplanting to the central flower for about 52 days, and the growth period of the field is about 125 days. Yunyan 97 is an important main cultivation flue-cured tobacco variety in tobacco production in China in recent years and also an important industrial common variety in cigarette production.
Single Nucleotide Polymorphism (SNP) mainly refers to a DNA sequence polymorphism caused by a single nucleotide variation on a genome. Compared with the traditional molecular marker, the SNP marker has the advantages of high density, wide distribution range, simple typing and the like. With the development of whole genome sequence identification technology and automated SNP chip typing technology, genetic diversity studies using large-scale, high-throughput SNP chips have become increasingly common. The national Tobacco gene research center designs and develops the first Tobacco high-density SNP chip (420K Tobacco SNP array), covers most of labeled SNP sites and uniformly distributes the whole Tobacco genome, and provides convenience for genetic diversity research on Tobacco varieties on the whole genome level. The genetic diversity of tobacco main cultivars is analyzed by adopting a tobacco high-density SNP chip marking technology, and specific SNP molecular markers of specific main cultivars can be obtained by screening. The MassARRAY molecular weight array platform based on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology can design up to 40 PCR reactions and genotype detection aiming at SNP sites, and has very flexible experimental design and high typing result accuracy. According to application requirements, when hundreds to thousands of samples are detected for dozens to hundreds of SNP sites, MassARRAY has extremely high cost performance. Is particularly suitable for large-scale typing detection of a limited number of SNP sites.
The variety is the basis of the production of high-quality tobacco leaf raw materials and is one of the most main factors influencing the quality of the tobacco leaves. According to the seed Law of the people's republic of China and the tobacco monopoly Law, in order to ensure the stability and sustainable development of tobacco production, a variety approved (approved) by the national committee for examining and determining tobacco varieties must be selected, and inferior and impure varieties are strictly forbidden to be planted. In addition, tobacco industry production and product development also impose stringent requirements on specific tobacco leaf varieties. At present, China faces the problems that the genetic background of tobacco main cultivars is narrow, and the similarity degree on morphology is high, so that the tobacco main cultivars are difficult to distinguish and identify. The detection method of tobacco varieties mainly comprises a field planting identification method. However, the method has the defects of large field planting scale, multiple identification items (identification characters relate to plant height, leaf number, pitch, leaf length, leaf width, stem leaf angle and the like), long period (spanning different growth periods of tobacco), large identification difficulty (character index difference is small), difference (influenced by environment) between years with the same character and the like. The DNA identity identification of tobacco is urgently needed from the molecular level in the aspects of tobacco variety protection, seed purity monitoring, tobacco true and false detection and the like.
Patent CN1057234141A discloses a molecular biological method for identifying the purity of tobacco varieties, which comprises: respectively extracting the total genomic DNA of the control tobacco variety and the tobacco variety to be detected, performing whole genome sequencing on the control tobacco variety and the tobacco variety to be detected at a proper depth by adopting the latest sequencing technology, performing sequence splicing, assembling and whole genome sequence comparison on the control tobacco variety and the tobacco variety by utilizing a bioinformatics means based on a tobacco genome reference sequence, counting the base difference of the control tobacco variety and the tobacco variety to be detected, and calculating the purity percentage of the control tobacco variety. The method is not influenced by environmental conditions and seasons, has accurate and reliable identification result, can accurately identify the purity of the tobacco variety from the single base variation level of the minimum genetic unit, and is used for the purification and authenticity identification of the tobacco variety parents. However, this method is complicated to operate and very expensive to detect.
Disclosure of Invention
The invention aims to provide a primer combination for identifying flue-cured tobacco yunyan 97, which can simply, quickly and efficiently identify whether a detection sample is the flue-cured tobacco yunyan 97.
Another object of the present invention is to provide an identification kit comprising the above primer combination.
The invention also provides application of the primer combination and the identification kit.
The invention also provides a method for simply, quickly and efficiently identifying the flue-cured tobacco yunyan 97.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the primer combination is used for identifying the flue-cured tobacco yunyan 97, the primers are respectively designed aiming at 13 specific SNP locus flanking sequences of the flue-cured tobacco yunyan 97, and the primers are specifically designed according to different corresponding detection methods. The 13 SNP markers are specific SNP sites which are selected based on the whole genome SNP typing results of main tobacco cultivars in China in recent years and are unique to flue-cured tobacco Yunyan 97, the physical positions of the specific SNP sites are determined based on whole genome sequence comparison of Hongda major cultivars in tobacco cultivation, and specific site information is shown in the following table 1 and is respectively named as FP01-FP 13. The gene sequence containing the 13 SNP loci is shown as SEQ ID NO 1-13.
TABLE 1 13 specific SNP sites of Yunyan 97 cured tobacco
The detection method can adopt SNP classical detection methods such AS PCR-RFLP, single-strand conformation polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE) and allele specific PCR (AS-PCR) or SNPs high-throughput detection methods such AS DNA sequencing method, gene chip technology, Denaturing High Performance Liquid Chromatography (DHPLC), Taqman probe method, SNap shot method and MassARRAY molecular weight array technology to realize variety identification and breeding material detection of flue-cured tobacco Yunyan 97.
The flue-cured tobacco Yunyan 97 identification kit can also comprise PCR buffer solution and MgCl besides the primer combination2dNTPs, PCR Enzyme, SAP buffer, iPLEX termination mix, iPLEX Enzyme, water, etc.
The primer combination or the kit is applied to the identification of the flue-cured tobacco yunyan 97 variety. The method specifically comprises the following steps: and (3) taking the genomic DNA of the tobacco sample to carry out SNP typing detection, and if the genotypes of 13 SNP sites in the detected sample are completely consistent with the results shown in the following table 2, judging that the tobacco sample is flue-cured tobacco Yunyan 97.
TABLE 2 genotype of each SNP site in cured tobacco Yunyan 97
| FP01 | FP02 | FP03 | FP04 | FP05 | FP06 | FP07 | FP08 | FP09 | FP10 | FP11 | FP12 | FP13 |
| TT | AA | AA | AA | AA | AA | TT | AA | AA | TT | TT | CC | TT |
The invention preferably adopts a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology (MassARRAY molecular weight array platform), and designs two amplification primers and one extension primer aiming at each site based on the Assay Design Suit (Agena), wherein the primer combination is shown in the following table 3.
TABLE 3 primer combinations designed based on specific SNP sites of flue-cured tobacco Yunyan 97
The method for identifying the flue-cured tobacco yunyan 97 comprises the following steps:
1) SNP locus multiplex PCR amplification reaction
Taking the genome DNA of the tobacco sample as a template, and carrying out multiple PCR amplification reaction by using an amplification primer in the primer combination to obtain a PCR product;
2) SAP enzymatic reaction
Removing residual dNTP and primers in the PCR product by SAP enzyme to obtain a reaction product;
3) single base extension reaction
Adding an extension primer into the reaction product to carry out single base extension reaction to obtain an extension product;
4) genotype detection and result determination
And (3) preprocessing the extension product, carrying out SNP genotype detection by using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, and if the genotypes of the SNP markers FP01-FP13 in the detected sample are TT, AA, TT, AA, TT, CC and TT in sequence, judging that the tobacco sample is flue-cured tobacco cloud cigarette 97.
The multiplex PCR amplification reaction in the step 1) comprises the following steps: reaction system: 10 XPCR buffer 0.5. mu.L, 25mM MgCl2mu.L 0.4, 0.1. mu.L 25mM dNTPs, 0.2. mu.L 5U/. mu.L PCR Enzyme, 1. mu.L of a mixture of 1. mu.M amplification primers, 1. mu.L of 10 ng/. mu.L tobacco sample genomic DNA, and water to 5. mu.L; the reaction conditions are as follows: 2min at 95 ℃; 45 cycles of 95 ℃ for 30s, 56 ℃ for 30s, and 72 ℃ for 1 min; 5min at 72 ℃.
The SAP enzyme reaction in the step 2) is as follows: adding 0.3. mu.L SAP 1.7U/. mu.L and 0.17. mu.L SAP buffer 10 Xto the PCR product, and adding water to make up to 7. mu.L; the reaction conditions are as follows: at 37 ℃ for 40min and at 85 ℃ for 5 min.
The single base extension reaction in the step 3) is as follows: adding 0.2. mu.L of 10 xiPLEX buffer solution, 0.2. mu.L of 10 xiPLEX termination mix, 0.041. mu.L of 33U/. mu.L iPLEX Enzyme and 0.94. mu.L of 1. mu.M extension primer into the reaction product, and adding water to 9. mu.L; the reaction conditions are as follows: 30s at 94 ℃; 5 cycles of [94 ℃ for 5s, (52 ℃ for 5s, 80 ℃ for 5s) ] < 40 cycles; 3min at 72 ℃.
The pretreatment in the step 4) comprises the following steps: adding water 41 μ L and clean resin 15mg into the extension product, mixing, desalting, removing ions, and centrifuging to obtain supernatant.
The SNP genotype detection in the step 4) is as follows: and (3) using a MassARRAY Nanodispenser RS1000 spotting instrument to spot the supernatant onto a 384-spot SpectroCHIP chip, placing the chip into a MassARRAY type workbench MA4, and scanning the chip by using a MALDI-TOF (matrix assisted laser desorption ionization time of flight) mass spectrometer to obtain an SNP genotype detection result.
All primers in the primer mixture used in the present invention are equal in amount.
The invention has the beneficial effects that:
the invention utilizes a tobacco 420K high-density SNP chip to carry out whole genome SNP typing on main tobacco cultivars in China in recent years, obtains a set of 13 specific SNP markers suitable for identifying flue-cured tobacco cloud 97 by screening according to polymorphism SNP sites among varieties, the specific site information is shown in table 1, corresponding primers are designed according to the site information, the specific sequence is shown in table 3, a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology is utilized to carry out SNP typing detection on candidate samples, a set of simple, convenient, fast, efficient and reliable molecular detection system for the flue-cured tobacco cloud 97 cultivars is established, and a basis and basis are provided for the identification technical system for the flue-cured tobacco cloud 97.
The MassARRAY molecular weight array platform based on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology can be used for designing up to 40 PCR reactions and genotype detection aiming at SNP sites, and is very flexible in experimental design and high in typing result accuracy. According to application requirements, when hundreds to thousands of samples are detected for dozens to hundreds of SNP sites, MassARRAY has extremely high cost performance. Is particularly suitable for large-scale typing detection of a limited number of SNP sites.
The invention utilizes the high-density SNP chip of the tobacco 420K to carry out whole genome SNP typing on different flue-cured tobacco main cultivated varieties in China, and a set of specific SNP molecular markers suitable for identifying the flue-cured tobacco Yunyan 97 are obtained by screening. Comparing 13 SNP site flanking sequences obtained by cultivating a tobacco reference genome, designing a primer based on the SNP site flanking sequences, and performing typing detection on the sites by adopting a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology. And according to the SNP typing result obtained from the detection result, if the genotypes of the SNP markers FP01-FP13 in the detection sample are TT, AA, TT, AA, TT, CC and TT in sequence, judging that the tobacco sample is the flue-cured tobacco cloud tobacco 97. The method has the advantages of small sample dosage, short period, accurate identification result, good repeatability, high detection flux and good application and popularization prospects during detection.
Detailed Description
The present invention will be described in further detail with reference to specific examples. All primers in this example were synthesized from the Huada gene.
Example 1
The primer combination for identifying the flue-cured tobacco cloud 97 is designed aiming at the specific SNP locus marker of the flue-cured tobacco cloud 97, and comprises the following steps:
1. and (3) carrying out whole genome SNP typing detection on tobacco main cultivars.
420K Tobacco SNP array (Affymetrix) is used for carrying out whole genome scanning on Tobacco main cultivars in China in recent years, and SNP typing of sample DNA is carried out by relying on the Gene Titan chip platform (Affymetrix) of the national Tobacco Gene research center. The tobacco sample DNA is randomly fragmented into fragments of 25 to 125bp after genome-wide amplification. The fragments were purified and resuspended and hybridized with 420K Tobacco SNP array. Each SNP is differentially ligated by a two-color ligation reaction occurring on the surface of the chip. After completion of the hybridization process, stringent washes are performed to remove non-specific binding. And after the connection reaction is finished, the chip finishes the steps of dyeing and washing and the like on a Gene Titan multi-channel automatic chip workstation, and finally scanning and outputting the result. And processing the data obtained by the chip analysis to obtain SNP typing results of different varieties.
2. And (3) screening specific SNP sites of flue-cured tobacco yunyan 97.
According to the grading and recommendation types of the data of the chip system, only two types of site data of Poly high resolution and Mono high resolution are selected, high-quality SNP (single nucleotide polymorphism) typing results are obtained after filtering, sites with 100% of calling rates in all detected varieties are reserved, sites with heterozygous typing in any variety are further screened and removed, and finally, homozygous SNP sites in all detected varieties are obtained. And screening to obtain specific SNP sites in the flue-cured tobacco yunyan 97. Because a large number of repetitive sequences exist in tobacco, in order to avoid non-specific amplification in the design of detection primers, the blast comparison is carried out on 200bp sequences of the flanks of SNP sites and a reference genome, and sites without highly similar sequences in the genome are screened. Combining the distribution condition of SNP loci on a chromosome, selecting one locus on the chromosome with polymorphic loci, and preferably selecting two loci with chromosome polymorphism as specific SNP markers of flue-cured tobacco yunyan 97.
13 specific SNP molecular markers for identifying the flue-cured tobacco yunyan 97 are obtained by screening, the 13 SNP markers are unique specific SNP loci of the flue-cured tobacco yunyan 97 screened out based on the whole-genome SNP typing results of main tobacco cultivars in China in recent years, and the physical positions of the specific SNP loci are determined based on the whole-genome sequence comparison of the Honghuadajinyuan of the cultivated tobacco cultivars; the 13 SNP markers are FP01-FP13, and the specific site information is shown in the table 1.
3. And (3) designing a primer combination for identifying the flue-cured tobacco yunyan 97.
And carrying out chromosome positioning on the screened specific SNP sites in a reference genome to obtain an upstream sequence and a downstream sequence containing the SNP sites. Based on the Assay Design Suit (Agena), two amplification primers and one extension primer are designed for each site, and the primer sequences are shown in the invention content part of Table 3.
Example 2
Flue-cured tobacco yunyan 97 identification kit: comprises 5mL of the amplification primer mixture (1. mu.M) of example 1, 5mL of 10 XPCR buffer, and 25mM MgCl24mL, 25mM dNTPs 2mL, 5U/. mu.L PCR Enzyme 2mL, 1.7U/. mu.L SAP3mL, 10 XSAP buffer 3mL, 10 XPLEX buffer 2mL, 10 XPLEX termination mix 2mL, 33U/. mu.L iPLEX Enzyme 1mL, extension primer mix (1. mu.M) 5mL in example 1, and water 200 mL.
Example 3
The method for identifying the flue-cured tobacco yunyan 97 comprises the following steps:
1. extracting DNA of a sample to be detected: collecting fresh leaf tissue of a sample, and extracting Genomic DNA of the sample by utilizing a Gene Pure New way Plant Genomic DNA Kit (Gene Answer) Kit; the DNA concentration was measured by using a nucleic acid protein analyzer NanoDrop ND-2000(ThermoFisher Scientific), and the DNA was diluted to 10 ng/. mu.L for use.
2. MassARRAY detection: the operation method is carried out according to the requirements of a MassARRAY system platform (Agena), the reaction is carried out by utilizing an iPLEX Gold Reagent Kit (Agena), and the method specifically comprises the following steps:
1) SNP locus multiplex PCR amplification reaction:
taking the genome DNA of a sample to be detected as a template, and carrying out multiple PCR amplification reaction by using the amplification primers in the embodiment 1 to obtain a PCR product;
the reaction system is as follows: PC (personal computer)R buffer (10X) 0.5. mu. L, MgCl20.4. mu. L, dNTPs (25mM) 0.1. mu.L (25mM), PCR Enzyme (5U/. mu.L) 0.2. mu.L, 1. mu.L of mixture of amplification primers (1. mu.M), 1. mu.L of genomic DNA (10 ng/. mu.L), and water to make up to 5. mu.L. The reaction conditions are as follows: 2min at 95 ℃; 45 cycles of 95 ℃ for 30s, 56 ℃ for 30s, and 72 ℃ for 1 min; 5min at 72 ℃.
2) SAP enzyme reaction:
removing dNTPs from the PCR product by Shrimp Alkaline Phosphatase (SAP); adding SAP (1.7U/. mu.L) 0.3. mu. L, SAP buffer (10X) 0.17. mu.L to the PCR product in step 1), and adding water to make up to 7. mu.L; the reaction conditions are as follows: at 37 ℃ for 40min and at 85 ℃ for 5 min.
3) Single base extension reaction:
extension reactions were performed using the iPLEX Reagent Kit: adding 0.2 mu L of iPLEX buffer solution (10X), 0.2 mu L of iPLEX determination mix (10X), 0.041 mu L of iPLEX Enzyme (33U/. mu.L), 0.94 mu L of a mixture of (1 mu.M) extension primers and water to the product obtained in the step 2) to make up to 9 mu L; the reaction conditions are as follows: 30s at 94 ℃; 5 cycles of [94 ℃ for 5s, (52 ℃ for 5s, 80 ℃ for 5s) ] < 40 cycles; 3min at 72 ℃;
4) and (3) genotype detection:
adding 41 mu L of water and 15mg of clean resin (96-hole plate) into the product obtained in the step 3), and carrying out desalting, deionization and interference prevention treatment by reversing and shaking for 15 min; centrifuging at 3200g for 5 min; and taking the supernatant for later use. Spotting the sample on 384-spot SpectroCHIP (chip) using a MassARRAY Nanodispenser RS1000 spotter on the supernatant of the extension product of step 4). The chip was placed in a MassARRAY type workbench MA4, the chip was scanned using a MALDI-TOF (matrix assisted laser Desorption ionization time of flight) mass spectrometer, the scan was analyzed with the type 4.0 software and the results were derived.
3. Comparison of the results
And judging the obtained SNP marker detection result, and if the detection results of 13 SNP sites in the detection sample are completely consistent with the Yunyan tobacco 97 fingerprint result, determining that the variety to be identified is the flue-cured tobacco Yunyan tobacco 97.
The MassARRAY molecular weight array platform based on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology can design 13-fold PCR reaction and genotype detection aiming at 13 SNP sites, and has flexible experimental design and high accuracy of typing results. 396 samples can be simultaneously detected for 13 SNP loci, and the detection flux is high.
Test examples
The method of example 3 was used to identify 24 samples of different species, and the results of the method were shown in Table 4. The samples 1 to 24 involved in the detection are respectively: flue-cured tobacco K326, safflower Dajinyuan, Zhongyan 100, Cuibi No. 1, Yunyan 85, Yunyan 87, Yunyan 97, Yunyan 100, NC95, Longjiang 911, Longjiang 981, Qin tobacco 96, Bina No. 1 and Nanjiang No. 3; aromatic cigarettes, Yunxiang Basman No. 1, Yunxiang No. 2 and Basma; BAIBAIYAN 'EYEYAN No. 1, BAIBAIYAN' 3, VAM, Burley-21; cigar Beiinhart-1000, Havana-10, Florida-301. Participating in detection of flue-cured tobacco K326, Honghuadajinyuan, Zhongyan 100, Cuibi No. 1, Yunyan 85, Yunyan 87, Yunyan 97, Yunyan 100, Longjiang 911, Longjiang 981, Qin tobacco 96, Bina No. 1 and Nanjiang No. 3; aromatic cigarettes, Yunxiang Basman No. 1 and Yunxiang No. 2; Bai-Ri-Yan Hu-Yan No. 1 and Hu-Yan No. 3 are the main cultivated varieties which are popularized and used in the tobacco production of China in recent years.
TABLE 424 tobacco sample test results
| FP01 | FP02 | FP03 | FP04 | FP05 | FP06 | FP07 | FP08 | FP09 | FP10 | FP11 | FP12 | FP13 | |
| Sample 1 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 2 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 3 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample No. 4 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample No. 5 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample No. 6 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 7 | TT | AA | AA | AA | AA | AA | TT | AA | AA | TT | TT | CC | TT |
| Sample 8 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 9 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 10 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 11 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 12 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 13 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 14 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 15 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 16 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 17 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 18 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 19 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 20 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 21 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 22 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 23 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 24 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
As can be seen from Table 4, the SNP site detection result of only sample 7 in 24 samples is completely consistent with the fingerprint result of Yunyan cigarette 97, which indicates that the detection method has specificity to the flue-cured tobacco Yunyan cigarette 97.
<110> Zhengzhou tobacco institute of China tobacco general Co
<120> primer combination and kit for identifying flue-cured tobacco yunyan 97, application and detection method
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<170> SIPOSequenceListing 1.0
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<221> Gene sequence comprising FP01
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agcctaccgc atttcctgtt agcacattca ttcatccttg tgtcatatct acatactgat 60
gcatattaag gtgtgaatag gatattaccc agccatttta ngcaactttc cacattgtca 120
tttactcgtt acttgtaccc cctactagat gttataacct agtcaatatg cttactgaaa 180
gatataccat tttttagagg a 201
<211> 201
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<221> Gene sequence comprising FP02
<400> 2
tgaggtcccg ggggcctcgg ggtgatttcg gagagttgtc ggaagaattg gaagtggttt 60
gaagcagctg aagctgctga tctctatcat aaccacacct ntggttgggt ctcgtagctg 120
tggagccgca gaagcagcgg agtcatcaca gaagcggaaa ggggagatgc cagcagggac 180
cgcagaagcg ggagcatacc a 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP03
<400> 3
actcaaagtt tttactattc ttgttttcct tcaaaaaatc cctctgcttc acaatctccg 60
catccaagtt ccttgagcaa atcatgtatc ttcttctaat ntttcttccc ttgttctcca 120
atggctaaaa catccaaaat cgtgccgcaa aaagaaactg cgtcttcgtc aaggcctgtc 180
ggcgtcggag ctgcggcaga a 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP04
<400> 4
ttgccttaat tgtagtcatt ttctcagtca tgatgttaca tccgtgtatt atatctctgt 60
ctatgtgcat tctttaatac gtttcccaca tgataatgat ntccttatat gtgtgacagg 120
gttcgagctg agtattgtga tatgtggaca tttgagactt gagctgttga tgattgatct 180
tgggccatag agccgatttg a 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP05
<400> 5
ttgaggtcat cctgggcatg gactggttat ccttatatca cgtcatcctt gattgtcatg 60
ccaagatagt tacgttagaa atgtcagagt tctcaagatt ngagtggaag ggttcctcca 120
ttagtacatc tagtcgggtt atctctttta tgaaggctct acatatagtc gagaagggtt 180
gtttggctta tctagcttat g 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP06
<400> 6
gtagcccggt ccggatttcg gagggtccat tcttctatct ttataacccg accctatctc 60
tataaataga cgtaagggtc catttttaag gggaatatct natagtttta gagagaggaa 120
gtgccctaga gtgagagtag aaatccttaa gctaattgtt catcaaaatc ttgctcaagc 180
cttgaaattc aacaagaaag c 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP07
<400> 7
aaccctcatt ggaagtcgcc tgatcacagc ctcttgtgtt atctttccat cccagctata 60
ttgcatgtca agtgtcttct tcttactttt gtttgaacca nttctcaagt atgtcggtga 120
aagatctcct ttcctagcta cgattgtaaa ggctgcatct agcatttctt cctcgtcccc 180
ttcttcgtag aaaccttctc c 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP08
<400> 8
ttcttattta tggccattag tgtagccttt aatggatatg atatttcttc attaggtgtt 60
tattctttat tattaaactg gcatacgtta atttgaactc ngtttgtatc aaagtacgct 120
ctttactctc tctagaacag cggtaactaa cggctacccc ctagcgagta gccacacgcg 180
cccccactat ggatccacca g 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP09
<400> 9
gaagaaagcc aaccgttgta gtcaatgcgg aaaccggcag gaacaacgcg catggtatgt 60
cttggcatct cgtacccatt tgaaagaaca ttgaaggaag naggagttca ggatcgtttc 120
ttatcatttc ttgccgcttt cactctatga aacatggaga ctatctgtat acggcaaatt 180
cggggccccg atttccccgt c 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP10
<400> 10
gtggatttca gagattaatc atctaatatt gatgtcttaa tggcctcacc atcatcatct 60
tgacaaaact tcacatctag tttcagtttt atcaaagttt ntacggcttg cttctctcag 120
gtacctaaag gatgggacaa actttccatc tctcttatct gcgtggaaac agggaaaaca 180
gtcagcaaat cagggaaagc c 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP11
<400> 11
agcatcaaat aatgccgtac gagacaaacc cggtcgataa aggtcgaatc tttaatcaaa 60
acgcaaagtc aaccagaaag gcaaacccat gaccgcacct nagtacccca caaaacttat 120
aaattccgat aacccattca attatgagtc caaccatact aatttcactc aaatccaact 180
ccgaatcgat gttcaaaact c 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP12
<400> 12
gtgagtccaa ctatgcaaaa atgaaatttg atagaatgtg gttccaaatt tatcataaaa 60
atatacatta aaaggaaagc atttgctgtt ggttaacacg nccgatcagt ttgttgctca 120
gttgcatgaa ctttaaatct ggtgtacaac attcctaata atgagagcgt ttgctaaaga 180
tggttaggta gaatggaagt a 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP13
<400> 13
ctaccttcaa agcaacaaca tgttcattca atccggacaa ctctgcctcc agatcttgga 60
tccgctcatc taatctcact accttgagca ccgacatctc nacttcaata tctcgctcag 120
acctacaaac acggagagca tcatctaggg ctgctgcttc agatatggca gccaccctgc 180
ctttctcggc acgagccaat t 201
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP01-F
<400> 14
acgttggatg gaataggata ttacccagcc 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP01-R
<400> 15
acgttggatg ggttataaca tctagtaggg 30
<211> 21
<212> DNA
<213> Artificial sequence
<221> FP01-E
<400> 16
ggatattacc cagccatttt a 21
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP02-F
<400> 17
acgttggatg tgaagctgct gatctctatc 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP02-R
<400> 18
acgttggatg ttccgcttct gtgatgactc 30
<211> 22
<212> DNA
<213> Artificial sequence
<221> FP02-E
<400> 19
catctctatc ataaccacac ct 22
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP03-F
<400> 20
acgttggatg aatctccgca tccaagttcc 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP03-R
<400> 21
acgttggatg ttgcggcacg attttggatg 30
<211> 18
<212> DNA
<213> Artificial sequence
<221> FP03-E
<400> 22
ggagaacaag ggaagaaa 18
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP04-F
<400> 23
acgttggatg atctctgtct atgtgcattc 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP04-R
<400> 24
acgttggatg acaatactca gctcgaaccc 30
<211> 22
<212> DNA
<213> Artificial sequence
<221> FP04-E
<400> 25
gggtttccca catgataatg at 22
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP05-F
<400> 26
acgttggatg gtcatgccaa gatagttacg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP05-R
<400> 27
acgttggatg cccgactaga tgtactaatg 30
<211> 15
<212> DNA
<213> Artificial sequence
<221> FP05-E
<400> 28
ggaacccttc cactc 15
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP06-F
<400> 29
acgttggatg ttataacccg accctatctc 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP06-R
<400> 30
acgttggatg tctagggcac ttcctctctc 30
<211> 21
<212> DNA
<213> Artificial sequence
<221> FP06-E
<400> 31
acttcctctc tctaaaacta t 21
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP07-F
<400> 32
acgttggatg tcccagctat attgcatgtc 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP07-R
<400> 33
acgttggatg ggaaaggaga tctttcaccg 30
<211> 18
<212> DNA
<213> Artificial sequence
<221> FP07-E
<400> 34
caccgacata cttgagaa 18
<211> 29
<212> DNA
<213> Artificial sequence
<221> FP08-F
<400> 35
acgttggatg attattaaac tggcatacg 29
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP08-R
<400> 36
acgttggatg gttaccgctg ttctagagag 30
<211> 19
<212> DNA
<213> Artificial sequence
<221> FP08-E
<400> 37
agcgtacttt gatacaaac 19
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP09-F
<400> 38
acgttggatg tggcatctcg tacccatttg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP09-R
<400> 39
acgttggatg catagagtga aagcggcaag 30
<211> 17
<212> DNA
<213> Artificial sequence
<221> FP09-E
<400> 40
aacgatcctg aactcct 17
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP10-F
<400> 41
acgttggatg gcctcaccat catcatcttg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP10-R
<400> 42
acgttggatg ccatccttta ggtacctgag 30
<211> 17
<212> DNA
<213> Artificial sequence
<221> FP10-E
<400> 43
gagagaagca agccgta 17
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP11-F
<400> 44
acgttggatg agaaaggcaa acccatgacc 30
<211> 31
<212> DNA
<213> Artificial sequence
<221> FP11-R
<400> 45
acgttggatg gttggactca taattgaatg g 31
<211> 20
<212> DNA
<213> Artificial sequence
<221> FP11-E
<400> 46
ggggacccat gaccgcacct 20
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP12-F
<400> 47
acgttggatg aggaaagcat ttgctgttgg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP12-R
<400> 48
acgttggatg agttcatgca actgagcaac 30
<211> 16
<212> DNA
<213> Artificial sequence
<221> FP12-E
<400> 49
gctgttggtt aacacg 16
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP13-F
<400> 50
acgttggatg ggatccgctc atctaatctc 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP13-R
<400> 51
acgttggatg tctccgtgtt tgtaggtctg 30
<211> 18
<212> DNA
<213> Artificial sequence
<221> FP13-E
<400> 52
tgagcgagat attgaagt 18
Claims (10)
1. The primer combination for identifying the flue-cured tobacco yunyan 97 is characterized in that the primer is designed aiming at 13 specific SNP site flanking sequences of the flue-cured tobacco yunyan 97, the site gene sequence containing 13 SNPs is shown as SEQ ID NO. 1 ~ 13, and n of the sequence SEQ ID NO. 1 ~ 13 is T, A, A, A, A, A, T, A, A, T, T, C, T in sequence.
2. The primer combination of claim 1, wherein the primer sequences of the 13 specific SNP sites are shown as SEQ ID NO. 14 ~ 52.
3. Flue-cured tobacco cloud 97 identification kit comprising the primer combination of claim 1 or 2.
4. The primer combination of claim 1 or 2 and the kit of claim 3 are used for identifying the variety of flue-cured tobacco yunyan 97.
5. The application of claim 4, which comprises the steps of taking genomic DNA of a tobacco sample to carry out SNP typing detection, judging the tobacco sample to be flue-cured tobacco cloud tobacco 97 if the genotypes of 13 SNP sites in the detected sample are completely consistent with those of flue-cured tobacco cloud tobacco 97, and sequentially carrying out TT, AA, TT, AA, TT, CC and TT on the genotypes of 13 SNP sites FP01 ~ FP13 in the flue-cured tobacco cloud tobacco 97.
6. The method for identifying the flue-cured tobacco cloud 97 by adopting the primer combination as claimed in claim 2, is characterized in that: the method comprises the following steps:
1) SNP locus multiplex PCR amplification reaction
Taking the genome DNA of the tobacco sample as a template, and carrying out multiple PCR amplification reaction by using an amplification primer in the primer combination to obtain a PCR product;
2) SAP enzymatic reaction
Removing residual dNTP and primers in the PCR product by SAP enzyme to obtain a reaction product;
3) single base extension reaction
Adding an extension primer into the reaction product to carry out single base extension reaction to obtain an extension product;
4) genotype detection and result determination
And (3) pretreating the extension product, carrying out SNP genotype detection by using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, and if the genotype of the SNP marker FP01 ~ FP13 in the detected sample is TT, AA, TT, AA, TT, CC and TT in sequence, judging that the tobacco sample is flue-cured tobacco cloud cigarette 97.
7. The method of claim 6, wherein: the multiplex PCR amplification reaction in the step 1) comprises the following steps: reaction system: 10 XPCR buffer 0.5. mu.L, 25mM MgCl2mu.L 0.4, 0.1. mu.L 25mM dNTPs, 0.2. mu.L 5U/. mu.L PCR Enzyme, 1. mu.L of a mixture of 1. mu.M amplification primers, 1. mu.L of 10 ng/. mu.L tobacco sample genomic DNA, and water to 5. mu.L; the reaction conditions are as follows: 2min at 95 ℃; 45 cycles of 95 ℃ for 30s, 56 ℃ for 30s, and 72 ℃ for 1 min; 5min at 72 ℃.
8. The method of claim 7, wherein: the SAP enzyme reaction in the step 2) is as follows: adding 0.3. mu.L SAP 1.7U/. mu.L and 0.17. mu.L SAP buffer 10 Xto the PCR product, and adding water to make up to 7. mu.L; the reaction conditions are as follows: at 37 ℃ for 40min and at 85 ℃ for 5 min.
9. The method of claim 8, wherein: the single base extension reaction in the step 3) is as follows: adding 0.2. mu.L of 10 xiPLEX buffer solution, 0.2. mu.L of 10 xiPLEX termination mix, 0.041. mu.L of 33U/. mu.L iPLEX enzyme and 0.94. mu.L of 1. mu.M extension primer into the reaction product, and adding water to 9. mu.L; the reaction conditions are as follows: 30s at 94 ℃; 5 cycles of 94 ℃ for 5s, 52 ℃ for 5s, 80 ℃ for 5s, 40 cycles; 3min at 72 ℃.
10. The method of claim 6, wherein: the pretreatment in the step 4) comprises the following steps: adding water 41 μ L and clean resin 15mg into the extension product, mixing, desalting and deionizing, centrifuging, and collecting supernatant; and (3) spotting the supernatant on the chip by using a spotting instrument, and scanning the chip by using a MALDI-TOF mass spectrometer to obtain an SNP genotype detection result.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103773866A (en) * | 2014-01-15 | 2014-05-07 | 湖南农业大学 | Molecular marker, primer, kit and detection method for rapidly detecting flue-cured tobacco scent |
| CN104726564A (en) * | 2015-02-15 | 2015-06-24 | 西南大学 | Primer set, kit and method for identifying different tobacco varieties |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103773866A (en) * | 2014-01-15 | 2014-05-07 | 湖南农业大学 | Molecular marker, primer, kit and detection method for rapidly detecting flue-cured tobacco scent |
| CN104726564A (en) * | 2015-02-15 | 2015-06-24 | 西南大学 | Primer set, kit and method for identifying different tobacco varieties |
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| Title |
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| 一种新的基于单碱基延伸的SNP芯片技术;董园园等;《遗传》;20090415;439―444 * |
| 利用基因组简约法开发烟草SNP 标记及遗传作图;肖炳光等;《作物学报》;20140116;397−404 * |
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