CN107354203B - Primer combination and kit for identifying cured tobacco Bina No. 1, application and detection method - Google Patents
Primer combination and kit for identifying cured tobacco Bina No. 1, application and detection method Download PDFInfo
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Abstract
The invention relates to a primer combination and a kit for identifying cured tobacco tona No. 1, an application and a detection method, 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 15 specific SNP markers suitable for identifying cured tobacco tonna No. 1 by screening according to polymorphism SNP sites among varieties, compares 15 SNP site flanking sequences obtained by cultivating a tobacco reference genome, and designs a primer based on the sequences, wherein the sequences are shown as SEQ ID NO. 1-15; 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. And obtaining an SNP typing result according to the detection result, and identifying whether the sample is Bina No. 1.
Description
Technical Field
The invention relates to a primer combination and a kit for identifying cured tobacco tona No. 1, an application and a detection method, 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 Bina 1 is a new cured tobacco variety which is bred from the natural variant strain of the cured tobacco variety Yunyan 85 by a system breeding method of Bijie city company of Guizhou tobacco company, and is approved by a Guizhou tobacco variety approval group of the national tobacco variety approval Committee in 10 months in 2012. The plant type is tower-shaped, the plant type is barrel-shaped after topping, the natural plant height is about 210cm, the topping plant height is about 150cm, the natural leaf number is about 32, the number of picked leaves is 24-26, the stem circumference is 9-10 cm, the pitch is 4.5cm, the waist leaf length is 78.2cm, the width is 29.4cm, the top leaf length is 61.5cm, and the width is 21.7 cm; the leaf shape is oblong, the leaf color is green, the leaf ears are large, the leaf tips are gradually sharp, and the flower crown is pink; the field growth is regular, the growth potential is strong, the tobacco leaves are good in layering and yellowing, and the tobacco leaves are easy to bake; transplanting to the central flower for about 72 days, and the growth period of the field is about 135 days. Bina No. 1 is an important main cultivation flue-cured tobacco variety in tobacco production in China in recent years, and is 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 CN105734141A 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 cured tobacco tona 1, which can simply, quickly and efficiently identify whether a detection sample is cured tobacco tona 1.
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 cured tobacco tona No. 1.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the primer combination is used for identifying cured tobacco Pita 1, the primers are respectively designed aiming at the flanking sequences of 15 specific SNP sites of cured tobacco Pita 1, and the primers are specifically designed according to different corresponding detection methods. The 15 SNP markers are specific SNP sites selected based on the whole genome SNP typing results of main tobacco cultivars in China in recent years, the physical positions of the specific SNP sites are determined based on whole genome sequence comparison of Honghuadajinyuan of cultivated tobacco cultivars, and the specific site information is shown in the following table 1 and is respectively named as FP01-FP 15. The gene sequence containing the 15 SNP loci is shown as SEQ ID NO 1-15.
TABLE 1 15 specific SNP sites of cured tobacco Bina No. 1
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 Bina No. 1.
The flue-cured tobacco bium No. 1 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 No. 1 variety of cured tobacco Bina. 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 15 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 tona No. 1.
TABLE 2 genotypes of individual SNP sites in cured tobacco Bina No. 1
| FP01 | FP02 | FP03 | FP04 | FP05 | FP06 | FP07 | FP08 | FP09 | FP10 | FP11 | FP12 | FP13 | FP14 | FP15 |
| GG | AA | TT | GG | TT | CC | TT | AA | TT | TT | CC | TT | CC | AA | 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 the specific SNP site of cured tobacco Bina No. 1
The identification method of cured tobacco tona 1 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 utilizing a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, and if the genotypes of SNP markers FP01-FP15 in the detection sample are GG, AA, TT, GG, TT, CC, TT, AA, TT, CC, AA and TT in sequence, judging that the tobacco sample is flue-cured tobacco Bina No. 1.
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 tobacco main cultivars in China in recent years, obtains a set of specific SNP markers suitable for identifying cured tobacco Pitan 1 according to the inter-species polymorphism SNP site screening, the specific site information is shown in the table 1, corresponding primers are designed according to the site information, the specific sequence is shown in the table 3, the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology is utilized to carry out SNP typing detection on candidate samples, a simple, convenient, fast, efficient and reliable molecular detection system for cured tobacco Pitan 1 variety is established, and a basis and basis are provided for the identification technology system for cured tobacco Pitan 1.
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 flue-cured tobacco tona No. 1 are obtained by screening. Comparing and cultivating 15 SNP site flanking sequences obtained by 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 (3) according to the SNP typing result obtained from the detection result, if the genotypes of the SNP markers FP01-FP15 in the detection sample are GG, AA, TT, GG, TT, CC, TT, AA, TT, CC, TT, AA and TT in sequence, judging that the tobacco sample is flue-cured tobacco Bina No. 1. 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 cured tobacco bina No. 1 is designed aiming at specific SNP locus markers of cured tobacco bina No. 1 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 cured tobacco tona No. 1.
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 the specific SNP locus in flue-cured tobacco Bina No. 1. 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 sites on a chromosome, selecting one site on the chromosome with polymorphic sites, and selecting two sites with better chromosome polymorphism as specific SNP markers of flue-cured tobacco Bina No. 1.
The method obtains 15 specific SNP molecular markers for identifying flue-cured tobacco tona No. 1 by screening, wherein the 15 SNP markers are unique specific SNP loci selected based on the whole-genome SNP typing result of Chinese tobacco main cultivars in recent years, and the physical positions of the specific SNP loci are determined based on the whole-genome sequence comparison of cultivated tobacco cultivars Hongda large gold dollars; the 15 SNP markers are FP01-FP15, and the specific site information contents are shown in Table 1.
3. And (3) primer combination design for identifying flue-cured tobacco Pita 1.
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 tona No. 1 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 identification method of cured tobacco tona 1 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: PCR 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 1 mu M extension primer mixture 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 15 SNP sites in the detection sample are completely consistent with the fingerprint result of the Bina No. 1, determining that the variety to be identified is the cured tobacco Bina No. 1.
The MassARRAY molecular weight array platform based on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology can design 15-fold PCR reaction and genotype detection aiming at 15 SNP sites, and is flexible in experimental design and high in typing result accuracy. 396 samples can be simultaneously detected for 15 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 | FP14 | FP15 | |
| Sample 1 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 2 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 3 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample No. 4 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample No. 5 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample No. 6 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 7 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 8 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 9 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 10 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 11 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 12 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 13 | GG | AA | TT | GG | TT | CC | TT | AA | TT | TT | CC | TT | CC | AA | TT |
| Sample 14 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 15 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 16 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 17 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 18 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 19 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 20 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 21 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 22 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 23 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 24 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
As can be seen from Table 4, the result of the SNP site detection of only sample 13 in 24 samples is completely consistent with the result of the fingerprint of Bina No. 1, which indicates that the detection method has specificity to cured tobacco Bina No. 1.
<110> Zhengzhou tobacco institute of China tobacco general Co
<120> primer combination and kit for identifying cured tobacco Pita 1, application and detection method
<160> 60
<170> SIPOSequenceListing 1.0
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP01
<400> 1
gcttatagat atggtataga gtgactaagg tacatgtaaa ggtggcatct ttttataagt 60
atattagtgc ttctataaat tctttcatct cccacatgtt nctttgtgac ttacctactc 120
aatacattct tccatactga cgtccccact gggaatgcta catttcatgt tgcaggcaca 180
gatactcaag ctagtagacc t 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP02
<400> 2
gacatccgcc catgtcatta tttggaaatc gagcaggctt tctttcaact ttcgggaggc 60
gtcgaagctc ctcggattca aaccctctgt gaatacctcg nctgcccatt gataagtgag 120
gattttgact gcttattagc accttttagc ttttatttta gtccaaaatc attgaattgt 180
gttcccgaaa ctaatgaaat t 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP03
<400> 3
agctcgggct cgacccaaac attgaccccg aagtccctca tcgaccagtc ttacacccgg 60
gaagtagggt ttctcggttt atttaactcc ctgattgagc ntgtatcctc tcgtaaaacg 120
cgcccgaaca gtaagccttt aggtttggtt atctccccat tcttagctcg catttcatca 180
ttaaacttca tattcttagc a 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP04
<400> 4
ttattaattt cacattctcg tattacctta gctatgatga ctctccctta tagttaaatg 60
atatttttgg catgttatac acttctataa tttaagaccg naagatccaa attaaaagta 120
gtgtttatat tcttacatgg tgtccagtca aactaccatg cctaaaatgt agtggagtga 180
gcaagatttc ataagaataa c 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP05
<400> 5
aataaagcac atcaaaaata gaacatatat caaaataagg catcatatgc cacaacttgc 60
caaaattaac aatactgaag ttatcaaaac atatatccca ngactcaacc acaatacgaa 120
caagaatctc aataataaca aaatgaacgt gaattactaa acaaggaaag atatctcggt 180
aatcaataac ttcaaataag a 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP06
<400> 6
gaaaatgtat atatactttt gtggtatgat atcactggga tgttggggtt tgcatttaaa 60
gtcattcatt ggcacttaga cctgtaggga agattaagcg ngagtggtta ggtgtatttg 120
ggagtagatt ttagttcctg gttgggttat tctccctgac acaagcactg ccctgggccc 180
tgagaccctt gggaactttg a 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP07
<400> 7
aggtggttat cagaactgta tctatgcttt agcggatcga caggactagg agtcttcacc 60
agacgttgtg ataggtatat tgacaatttg ctcttatgat ncttatacct tgatggatcc 120
aggatctaat ttatcgtata ttaccccatt tgtcgtgggg aagtttggta tagtgcgtgg 180
aattttatgt tatccctttg t 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP08
<400> 8
atctcttcga gggtctttct tcgagatttg cagcgagcat attcttcaac cctccctttg 60
cggtcacgga tccttctcag ctcagcttga gcatcagcag natcctttaa atagattgac 120
atctcccggt gtgccttagt ccgacttatc gctgcttttg cacgagcatc aacgatctca 180
accctgattt ttgaaagctc g 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP09
<400> 9
ggaccgcggt agcttgaatt ccaaaaatga caactctctg aatccttacc accaaagacc 60
gcgataaaag gattgtggtt gtggtgggtc ttctacgtcc ntggtggact ttccgttgta 120
gcgctgcttt gacttagttt tgaatttctg caggtttcac taattttttg agctggttat 180
gacatccttg tccctttttg c 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP10
<400> 10
atgtcattca ttttcattac caaaataaag acaactcctc aattctgaat aacaattttt 60
tctcttgtca agaaatcttg taatatctct acacttaaaa ntagaatgac aggtatctta 120
agcatttgga ttagcggaag taccgtaatt cccattttag gcagtatcgt ctcaataaaa 180
atacgggtct tcctgtaaca a 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP11
<400> 11
gtcatggtct taatttgttg ctaggttcaa aaggctctat acatgcactt aaacttcaat 60
agttagcctt tcctgttata gcttttgtga catccctaga nccattgcct tattcttgct 120
tagttcaaaa ggctcgataa tttcttttag acaggctcat gcattaaaag tccataagct 180
gttggattaa ccacaaattg a 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP12
<400> 12
tattatgcag ttgatattgt gctaagtatt gttctatcac agatggtgag aacgcatgcg 60
gcagatgtac caggcgatag atgagattct cccttcattg ntagaggacg aggtagaggc 120
cgggggaggg ctccagctcc tattagagga cgaaggcgtc ctagagttgc tcccgttgta 180
ccactagtgg atccaatgca g 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP13
<400> 13
gatcacaaca catgtacgag taatcaagtt gtctcacaat ccacctagcc taatagagta 60
ttacatggaa tagctgacga caggaataac attcaatgcc ngatgactca tccgtaagca 120
attctatgac gaatgaactc atccgaactg acgtagagca cacattcaca ttagaccaac 180
aaacggacct caaaccgatt c 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP14
<400> 14
ctgcatgtgt tgcttgattt gttgttactc ctaccccctt cttgaatttg agatctgcat 60
agtatccatg taacttgtaa caattgtcgc tagtgcatcc nttgaaatgc agtagtcgcg 120
ttgcaaattt ttcttcttct gcttgtaatt acgggggccg agtcctccta gtgtttcaga 180
agagtgttgt agcttcattc a 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP15
<400> 15
gagaacgaca acctcgaaaa ctatggagaa aatggtgtgg ttgttccagt tgttggcacg 60
ccaccacaga acccctataa cacacctgga ccgattccaa nagacgcgga tttgcaagac 120
gtgcaatagg tcgacgaaac ctcacatacc gacaggagca tacaccatgg cgaccaacag 180
gaagtccaaa gaaccccacc c 201
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP01-F
<400> 16
acgttggatg gtacatgtaa aggtggcatc 30
<211> 29
<212> DNA
<213> Artificial sequence
<221> FP01-R
<400> 17
acgttggatg tgtattgagt aggtaagtc 29
<211> 20
<212> DNA
<213> Artificial sequence
<221> FP01-E
<400> 18
tgagtaggta agtcacaaag 20
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP02-F
<400> 19
acgttggatg gaagctcctc ggattcaaac 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP02-R
<400> 20
acgttggatg aaaggtgcta ataagcagtc 30
<211> 17
<212> DNA
<213> Artificial sequence
<221> FP02-E
<400> 21
cctctgtgaa tacctcg 17
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP03-F
<400> 22
acgttggatg tcttacaccc gggaagtagg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP03-R
<400> 23
acgttggatg ctaaaggctt actgttcggg 30
<211> 18
<212> DNA
<213> Artificial sequence
<221> FP03-E
<400> 24
ttaactccct gattgagc 18
<211> 29
<212> DNA
<213> Artificial sequence
<221> FP04-F
<400> 25
acgttggatg ttggcatgtt atacacttc 29
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP04-R
<400> 26
acgttggatg tggtagtttg actggacacc 30
<211> 24
<212> DNA
<213> Artificial sequence
<221> FP04-E
<400> 27
ctacacttct ataatttaag accg 24
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP05-F
<400> 28
acgttggatg atcatatgcc acaacttgcc 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP05-R
<400> 29
acgttggatg gattcttgtt cgtattgtgg 30
<211> 23
<212> DNA
<213> Artificial sequence
<221> FP05-E
<400> 30
aagttatcaa aacatatatc cca 23
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP06-F
<400> 31
acgttggatg cacttagacc tgtagggaag 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP06-R
<400> 32
acgttggatg agggagaata acccaaccag 30
<211> 18
<212> DNA
<213> Artificial sequence
<221> FP06-E
<400> 33
tgtagggaag attaagcg 18
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP07-F
<400> 34
acgttggatg tcaccagacg ttgtgatagg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP07-R
<400> 35
acgttggatg tagatcctgg atccatcaag 30
<211> 21
<212> DNA
<213> Artificial sequence
<221> FP07-E
<400> 36
gtggatccat caaggtataa g 21
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP08-F
<400> 37
acgttggatg atccttctca gctcagcttg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP08-R
<400> 38
acgttggatg gataagtcgg actaaggcac 30
<211> 23
<212> DNA
<213> Artificial sequence
<221> FP08-E
<400> 39
agatgtcaat ctatttaaag gat 23
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP09-F
<400> 40
acgttggatg ccaaagaccg cgataaaagg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP09-R
<400> 41
acgttggatg agtcaaagca gcgctacaac 30
<211> 17
<212> DNA
<213> Artificial sequence
<221> FP09-E
<400> 42
ggacggaaag tccacca 17
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP10-F
<400> 43
acgttggatg ctcttgtcaa gaaatcttgt 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP10-R
<400> 44
acgttggatg tacggtactt ccgctaatcc 30
<211> 26
<212> DNA
<213> Artificial sequence
<221> FP10-E
<400> 45
atcttgtaat atctctacac ttaaaa 26
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP11-F
<400> 46
acgttggatg cctgttatag cttttgtgac 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP11-R
<400> 47
acgttggatg tgcatgagcc tgtctaaaag 30
<211> 21
<212> DNA
<213> Artificial sequence
<221> FP11-E
<400> 48
agcttttgtg acatccctag a 21
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP12-F
<400> 49
acgttggatg gcgatagatg agattctccc 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP12-R
<400> 50
acgttggatg gagcaactct aggacgcctt 30
<211> 17
<212> DNA
<213> Artificial sequence
<221> FP12-E
<400> 51
ctctacctcg tcctcta 17
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP13-F
<400> 52
acgttggatg atggaatagc tgacgacagg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP13-R
<400> 53
acgttggatg acgtcagttc ggatgagttc 30
<211> 25
<212> DNA
<213> Artificial sequence
<221> FP13-E
<400> 54
cacgccagga ataacattca atgcc 25
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP14-F
<400> 55
acgttggatg acccccttct tgaatttgag 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP14-R
<400> 56
acgttggatg aatttgcaac gcgactactg 30
<211> 20
<212> DNA
<213> Artificial sequence
<221> FP14-E
<400> 57
ccgcgactac tgcatttcaa 20
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP15-F
<400> 58
acgttggatg ccacagaacc cctataacac 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP15-R
<400> 59
acgttggatg gaggtttcgt cgacctattg 30
<211> 16
<212> DNA
<213> Artificial sequence
<221> FP15-E
<400> 60
cctggaccga ttccaa 16
Claims (10)
1. The primer combination for identifying cured tobacco tona No. 1 is characterized in that the primer is designed aiming at flanking sequences of 15 specific SNP sites of cured tobacco tona No. 1, the gene sequence containing 15 SNP sites is shown as SEQ ID NO. 1 ~ 15, and n of the sequence SEQ ID NO. 1 ~ 15 is G, A, T, G, T, C, T, A, T, T, C, T, C, A, T in sequence.
2. The primer combination of claim 1, wherein the primer sequences of the 15 specific SNP sites are represented by SEQ ID NO 16 ~ 60.
3. Cured tobacco pycnometer No. 1 identification kit comprising the primer combination according to claim 1 or 2.
4. The primer combination of claim 1 or 2 and the kit of claim 3 are applied to the identification of flue-cured tobacco variety Bina 1.
5. The application of claim 4 is characterized by comprising the steps of taking genomic DNA of a tobacco sample to carry out SNP typing detection, judging that the tobacco sample is flue-cured tobacco Bina 1 if the genotypes of 15 SNP sites in the detected sample are completely consistent with those of flue-cured tobacco Bina 1, and sequentially carrying out GG, AA, TT, GG, TT, CC, TT, AA, TT, CC, AA and TT on the genotypes of 15 SNP sites FP01 ~ FP15 in the flue-cured tobacco Bina 1.
6. The method for identifying cured tobacco tona 1 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) 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 genotype of the SNP marker FP01 ~ FP15 in the detection sample is GG, AA, TT, GG, TT, CC, TT, AA, TT, CC, AA and TT in sequence, judging that the tobacco sample is flue-cured tobacco Bina No. 1.
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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| 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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