CN107354206B - Primer combination and kit for identifying No. 3 of flue-cured tobacco Nanjiang, application and detection method - Google Patents
Primer combination and kit for identifying No. 3 of flue-cured tobacco Nanjiang, application and detection method Download PDFInfo
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Abstract
The invention relates to a primer combination and a kit for identifying No. 3 of Nanjiang flue-cured tobacco, an application and a detection method, and belongs to the technical field of tobacco variety identification. The invention utilizes the tobacco 420K high-density SNP chip to carry out whole genome SNP typing on main tobacco cultivars in China in recent years, and obtains a set of specific SNP markers suitable for identifying No. 3 of Nanjiang cured tobacco by screening according to polymorphic SNP sites among varieties, wherein the number of the specific SNP markers is 12; comparing 12 SNP locus flanking sequences obtained by cultivating a tobacco reference genome, wherein the sequences are shown as SEQ ID NO. 1-12, and designing a primer 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. And obtaining an SNP typing result according to the detection result, and identifying whether the sample is No. 3 of south river or not.
Description
Technical Field
The invention relates to a primer combination and a kit for identifying No. 3 of Nanjiang flue-cured tobacco, an application and a detection method, and belongs 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 Nanjiang No. 3 is a natural variant found from Honghuadajinyuan by tobacco company in Guiyang city, and is obtained through systematic breeding for many years. In 2009, the tobacco variety was approved by the national tobacco variety approval committee of Guizhou province. The plant shape is cylindrical, the field growth vigor is strong, the natural plant height is 150-; the color of the leaf is dark green, the leaf ear is small, the leaf tip is gradually sharp, the leaf surface is wrinkled, the leaf edge is wavy, the leaf shape is oval, the main pulse is in the middle, the angle of the stem and the leaf is in the middle, the hair is more, the axillary bud grows in the middle, the pitch is 4.8cm, and the stem circumference is 9.6 cm. Transplanting to the central flower for 70 days, and the growth period of the field is about 130 days. Nanjiang No. 3 is a main cultivation flue-cured tobacco variety in tobacco production in China in recent years.
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 No. 3 of flue-cured tobacco Nanjiang, which can simply, quickly and efficiently identify whether a detection sample is No. 3 of flue-cured tobacco Nanjiang.
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 No. 3 south China center of flue-cured tobacco.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the primer combination is used for identifying No. 3 of flue-cured tobacco Nanjiang, the primers are respectively designed aiming at 12 specific SNP site flanking sequences of No. 3 of flue-cured tobacco Nanjiang, and the primers are specifically designed according to different corresponding detection methods. The 12 SNP markers are specific SNP sites selected from flue-cured tobacco Nanjiang No. 3 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 12. The gene sequence containing the 12 SNP loci is shown as SEQ ID NO 1-12.
TABLE 1 flue-cured tobacco Nanjiang No. 3 12 specific SNP sites
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 the No. 3 of the south river of flue-cured tobacco.
The flue-cured tobacco Nanjiang No. 3 identification kit can also comprise a PCR buffer in addition to the primer combinationWashing solution, MgCl2dNTPs, PCR Enzyme, SAP buffer, iPLEX termination mix, iPLEX Enzyme, water, etc.
The primer combination or the kit is applied to the identification of No. 3 variety of flue-cured tobacco Nanjiang. 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 12 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 Nanjiang No. 3.
TABLE 2 genotype of each SNP site in Nanjiang No. 3 of flue-cured tobacco
| FP01 | FP02 | FP03 | FP04 | FP05 | FP06 | FP07 | FP08 | FP09 | FP10 | FP11 | FP12 |
| AA | GG | AA | GG | AA | TT | TT | AA | AA | AA | CC | GG |
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 combination designed based on specific SNP site of flue-cured tobacco Nanjiang No. 3
The flue-cured tobacco Nanjiang No. 3 identification 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 utilizing a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, and if the genotypes of the SNP markers FP01-FP12 in the detection sample are AA, GG, AA, TT, AA, CC and GG in sequence, judging that the tobacco sample is flue-cured tobacco Nanjiang No. 3.
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 cultivated varieties in China in recent years, obtains a set of specific SNP markers suitable for identifying No. 3 of the Nanjiang cured tobacco by screening polymorphic SNP sites among varieties, 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 set of simple, convenient, fast, efficient and reliable molecular detection system for No. 3 varieties of the Nanjiang cured tobacco is established, and a foundation and a basis are provided for the identification technology system for No. 3 of the Nanjiang cured tobacco.
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 No. 3 of Nanjiang cured tobacco is obtained by screening. Comparing 12 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 (3) according to the SNP typing result obtained from the detection result, if the genotypes of the SNP markers FP01-FP12 in the detection sample are AA, GG, AA, TT, AA, CC and GG in sequence, judging that the tobacco sample is flue-cured tobacco Nanjiang No. 3. 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 No. 3 of the Nanjiang flue-cured tobacco is designed aiming at the specific SNP locus marker of No. 3 of the Nanjiang flue-cured tobacco, 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 the cured tobacco Nanjiang No. 3.
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 the flue-cured tobacco Nanjiang No. 3. 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 selecting two loci with better chromosome polymorphism as specific SNP markers of the flue-cured tobacco Nanjiang No. 3.
The method obtains 12 specific SNP molecular markers for identifying No. 3 of Nanjiang flue-cured tobacco by screening, wherein the 12 SNP molecular markers are unique specific SNP loci of No. 3 of Nanjiang flue-cured tobacco screened based on the whole-genome SNP typing results of main tobacco cultivars of China in recent years, and the physical positions of the specific SNP loci are determined based on the whole-genome sequence comparison of Hongda major pivot of the cultivated tobacco cultivars; the 12 SNP markers are FP01-FP12, and the specific site information is shown in the table 1.
3. And (3) designing a primer combination for identifying No. 3 of Nanjiang flue-cured tobacco.
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 Nanjiang No. 3 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 SAP 3mL, 10 XSAP buffer 3mL, 10 XPLEX buffer 2mL, 10 XPLEX termination mix 2mL, 33U/. mu.L iLEX Enzyme 1mL, extension primer mix 1. mu.M 5mL in example 1, water 200 mL.
Example 3
The flue-cured tobacco Nanjiang No. 3 identification method 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 result of 12 SNP sites in the detection sample is completely consistent with the fingerprint result of Nanjiang No. 3, determining that the variety to be identified is flue-cured tobacco Nanjiang No. 3.
The MassARRAY molecular weight array platform based on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology can design 12-fold PCR reaction and genotype detection aiming at 12 SNP sites, and has flexible experimental design and high accuracy of typing results. 396 samples can be simultaneously detected for 12 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 | |
| Sample 1 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 2 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 3 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample No. 4 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample No. 5 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample No. 6 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 7 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 8 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 9 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 10 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 11 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 12 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 13 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 14 | AA | GG | AA | GG | AA | TT | TT | AA | AA | AA | CC | GG |
| Sample 15 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 16 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 17 | CC | GG | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 18 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 19 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 20 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 21 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 22 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 23 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
| Sample 24 | CC | TT | GG | AA | GG | CC | GG | CC | GG | CC | TT | TT |
As can be seen from Table 4, the SNP site detection result of only sample 14 in 24 samples is completely consistent with the fingerprint result of Nanjiang No. 3, which indicates that the detection method has specificity to Nanjiang No. 3 flue-cured tobacco.
<110> Zhengzhou tobacco institute of China tobacco general Co
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<221> Gene sequence comprising FP02
<400> 2
agattcttga agcattgaat aaaggggaga ttccatccac agggtccctt gtggaagtct 60
tcaacaaggg tattctggat cggtgtttga aactgtacaa ngagcggacg gctaacatgg 120
ttttaccaat gccaggggag tctttgcaaa aggttcatga agagcacaaa gaggcagcga 180
tgaacctctt tgatgaacag c 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP03
<400> 3
tcatttacat acattctcgt atacttgccc tatgtgtgag agttgtacta ttggcacgtg 60
agttgcccat gtggttataa gttattttta tcgctggcac ntgagttgtc cgcgcggata 120
tgagatattg ttgcttcctt agcctgtgaa ttgtccacgc agatatgagg tatcaatggt 180
attggcacgt gagttgtccg t 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP04
<400> 4
tatcttttgt caagagtaca agcctcaata tgtaaataag gagtcctccc caatctttct 60
ctattgctat tgtctttgca caagagtcat aaacagtact nttcatcttg ttttagaggc 120
tcaatactat atcctcctcc atgtactctt attttgtgat atatacatgt aggagactgc 180
ctaacccccc caccgtgaaa g 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP05
<400> 5
tggatattga caattaattc cacatacctg acagccacta aaactcagct tcctatcaat 60
atattcacat atcacatttg attcaaatct tatttcctgc naataacata atcggaaaaa 120
ttacacatca tttgttttga gttaaaaacc tggcatgcca taacgtttct tttgtttttg 180
gcttttgact actctgacaa g 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP06
<400> 6
aacgtgtcca taattaataa cgctttgttt atttattaag aatgttcggg ccaaagttgc 60
acgaacgcat actccggttt tattttaaag aaatcataac ngtgtcacgc gaacgtgtcc 120
acaaccacac taatatttta aacggcccta aaggtttttc tacgaacatt tataatttta 180
cctctacatt atgaaattaa c 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP07
<400> 7
gtggtgagct gtgaggttgt acgattaatc cccacgcatg ttctaatttt gttcaggcct 60
tatggcgatg cgggtgagat agccctcgtg atgttgattt nctcagtgca ccttatatat 120
acttgcttct ctcttatttt attacttcta ttcattgttc ccacaatttt acttgtgctc 180
tctgctgtgc ttgatatcag t 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP08
<400> 8
tccatatgga gccgagcaat gtgtattgcg atcctaggac gcaataagat gggattcatt 60
gaaggaacct gtaaaaagga aagttatggt ccaaatatga nagatctatg ggagagctgc 120
agtgctatcg ttctatcgtg gataatgaac tgtgtctcga aggacttatt aagtggagtt 180
atatattctt cagatgcatg t 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP09
<400> 9
acttgaacaa attacaattg gaacgctccg atctcgagat atggatcttt tggtaagatt 60
gcacagtgct gcaaagtcaa aatgctgaac atgtgtatct nttttaagaa catactgcag 120
ctaatcctta aaaaacatgg ccgtgatttt ttgatatgtt gcatctcttc gagttctgat 180
tttttaggta gaagtagttt g 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP10
<400> 10
attaatcatc ttggctatgc agatgatttg attcacttca cctctgggga tagaagataa 60
atcaaactta tcatgaggaa actcaatagg aactaacaag natcaagtca agcgataaac 120
aaagataaaa aaaatattct tgatccatga gaacaccaat agacgttcca atagaagaat 180
aaggaaatag acagggtacg a 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP11
<400> 11
cgaggaggat attaccggcc tcgcatggaa caagacgcca aagattttgt acgaaaatgt 60
gataagtgcc aacgctacgc atcacttgta tatcgagcga nagaaccctt acattaattt 120
ctgtccccat ggccattcat gaaatggggg atgggcatcg tcataccgct gccaccggct 180
cttgaaaagg taatgtttct t 201
<211> 201
<212> DNA
<213> sequence
<221> Gene sequence comprising FP12
<400> 12
tttttttttt ggtatgtcaa atcatcataa gcttttttct gcagatacac gaattttatc 60
tagtcttcct ccacatcatt ttaagcatgt ccagccttca nccacagggt caatttaagc 120
atgccaacta atacagtatt gggatatcat tgttatcatg ttgtgcattt ttcgcatctg 180
tgtaatatgt catctcatca a 201
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP01-F
<400> 13
acgttggatg aatcgcttta tgcgagaggg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP01-R
<400> 14
acgttggatg acttgtcaac actcgtccac 30
<211> 15
<212> DNA
<213> Artificial sequence
<221> FP01-E
<400> 15
cctccacgtc cacag 15
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP02-F
<400> 16
acgttggatg acaagggtat tctggatcgg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP02-R
<400> 17
acgttggatg tcccctggca ttggtaaaac 30
<211> 16
<212> DNA
<213> Artificial sequence
<221> FP02-E
<400> 18
ggttagccgt ccgctc 16
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP03-F
<400> 19
acgttggatg ccctatgtgt gagagttgta 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP03-R
<400> 20
acgttggatg tatctcatat ccgcgcggac 30
<211> 17
<212> DNA
<213> Artificial sequence
<221> FP03-E
<400> 21
atttttatcg ctggcac 17
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP04-F
<400> 22
acgttggatg agtcctcccc aatctttctc 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP04-R
<400> 23
acgttggatg ggaggatata gtattgagcc 30
<211> 20
<212> DNA
<213> Artificial sequence
<221> FP04-E
<400> 24
agcctctaaa acaagatgaa 20
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP05-F
<400> 25
acgttggatg gccactaaaa ctcagcttcc 30
<211> 29
<212> DNA
<213> Artificial sequence
<221> FP05-R
<400> 26
acgttggatg atgatgtgta atttttccg 29
<211> 24
<212> DNA
<213> Artificial sequence
<221> FP05-E
<400> 27
tgtaattttt ccgattatgt tatt 24
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP06-F
<400> 28
acgttggatg gaacgcatac tccggtttta 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP06-R
<400> 29
acgttggatg tagaaaaacc tttagggccg 30
<211> 19
<212> DNA
<213> Artificial sequence
<221> FP06-E
<400> 30
ccctacgttc gcgtgacac 19
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP07-F
<400> 31
acgttggatg gtgagatagc cctcgtgatg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP07-R
<400> 32
acgttggatg gtgggaacaa tgaatagaag 30
<211> 18
<212> DNA
<213> Artificial sequence
<221> FP07-E
<400> 33
ccctcgtgat gttgattt 18
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP08-F
<400> 34
acgttggatg ggattcattg aaggaacctg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP08-R
<400> 35
acgttggatg ccacgataga acgatagcac 30
<211> 22
<212> DNA
<213> Artificial sequence
<221> FP08-E
<400> 36
gaaagttatg gtccaaatat ga 22
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP09-F
<400> 37
acgttggatg ttgcacagtg ctgcaaagtc 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP09-R
<400> 38
acgttggatg cacggccatg ttttttaagg 30
<211> 22
<212> DNA
<213> Artificial sequence
<221> FP09-E
<400> 39
cccctgctga acatgtgtat ct 22
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP10-F
<400> 40
acgttggatg tgaggaaact caataggaac 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP10-R
<400> 41
acgttggatg tggtgttctc atggatcaag 30
<211> 21
<212> DNA
<213> Artificial sequence
<221> FP10-E
<400> 42
ttgtttatcg cttgacttga t 21
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP11-F
<400> 43
acgttggatg tgataagtgc caacgctacg 30
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP11-R
<400> 44
acgttggatg tcccccattt catgaatggc 30
<211> 23
<212> DNA
<213> Artificial sequence
<221> FP11-E
<400> 45
gcagaaatta atgtaagggt tct 23
<211> 30
<212> DNA
<213> Artificial sequence
<221> FP12-F
<400> 46
acgttggatg atctagtctt cctccacatc 30
<211> 31
<212> DNA
<213> Artificial sequence
<221> FP12-R
<400> 47
acgttggatg cccaatactg tattagttgg c 31
<211> 15
<212> DNA
<213> Artificial sequence
<221> FP12-E
<400> 48
catgtccagc cttca 15
Claims (10)
1. The primer combination for identifying the No. 3 of the flue-cured tobacco Nanjiang is characterized in that the primer is designed aiming at 12 specific SNP site flanking sequences of the No. 3 of the flue-cured tobacco Nanjiang, the gene sequence containing 12 SNP sites is shown as SEQ ID NO. 1 ~ 12, and n of the sequence SEQ ID NO. 1 ~ 12 is A, G, A, G, A, T, T, A, A, A, C, G in sequence.
2. The primer combination of claim 1, wherein the primer sequences of the 12 specific SNP sites are shown as SEQ ID NO. 13 ~ 48.
3. Flue-cured south-Jiang No. 3 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 applied to the identification of the variety Nanjiang No. 3 of flue-cured tobacco.
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 Nanjiang No. 3 if the genotypes of 12 SNP sites in the detected sample are completely consistent with those of flue-cured tobacco Nanjiang No. 3, and sequentially carrying out AA, GG, AA, TT, AA, CC and GG on the genotypes of 12 SNP sites FP01 ~ FP12 in the flue-cured tobacco Nanjiang No. 3.
6. The method for identifying cured tobacco Nanjiang No. 3 by adopting the primer combination as claimed in claim 2, which 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 ~ FP12 in the detection sample is AA, GG, AA, TT, AA, CC and GG in sequence, judging that the tobacco sample is flue-cured tobacco Nanjiang No. 3.
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 |
|---|---|---|---|---|
| CN102965443A (en) * | 2012-12-10 | 2013-03-13 | 山东农业大学 | Method for identifying purity of tobacco variety Zhongyan 90 by using specific molecular marker method |
| CN104726564A (en) * | 2015-02-15 | 2015-06-24 | 西南大学 | Primer set, kit and method for identifying different tobacco varieties |
| CN105734141A (en) * | 2016-03-31 | 2016-07-06 | 湖北省烟草科学研究院 | Molecular biology method for identifying purity of tobacco varieties |
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|---|---|---|---|---|
| CN102965443A (en) * | 2012-12-10 | 2013-03-13 | 山东农业大学 | Method for identifying purity of tobacco variety Zhongyan 90 by using specific molecular marker method |
| CN104726564A (en) * | 2015-02-15 | 2015-06-24 | 西南大学 | Primer set, kit and method for identifying different tobacco varieties |
| CN105734141A (en) * | 2016-03-31 | 2016-07-06 | 湖北省烟草科学研究院 | Molecular biology method for identifying purity of tobacco varieties |
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