CN107345252B - Primer combination and kit for identifying 96 of flue-cured tobacco Qin tobacco, application and identification method - Google Patents
Primer combination and kit for identifying 96 of flue-cured tobacco Qin tobacco, application and identification method Download PDFInfo
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Abstract
The invention discloses a primer combination and a kit for identifying 96 of flue-cured tobacco Qin tobacco, application and an identification method, belonging to the technical field of biomolecule identification. 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 specific SNP markers suitable for identifying 96 of flue-cured tobacco Qin tobacco according to the screening of polymorphism SNP loci among varieties, the physical positions of the specific SNP markers are determined based on the whole genome sequence comparison of the safflower large gold of the cultivated tobacco varieties, and the sequences containing the SNP loci are shown as SEQ ID NO: 1-12. Aiming at the SNP locus flanking sequence of the flue-cured tobacco Qin tobacco 96, corresponding primers are designed according to different detection methods, and the method can be used for variety identification of the flue-cured tobacco Qin tobacco 96. The invention establishes a set of flue-cured tobacco Qin tobacco 96 identification method based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, and has short detection period, high flux and accurate result.
Description
Technical Field
The invention relates to a primer combination for identifying 96 of flue-cured tobacco Qin tobacco, and also relates to a kit containing the primer combination, an application and an identification method, belonging to the technical field of biomolecule identification.
Background
Tobacco is an important economic crop and is a main raw material for cigarette production. The cured tobacco variety Qinyan 96 is obtained by hybridization breeding by using Shaanxi province tobacco research institute and Qingzhou tobacco research institute of China tobacco general company G-28 as a female parent and using leaf yellow as a male parent, and is approved by the national tobacco variety approval committee in 2009 at 3 months and 10 days. 96 flue-cured tobacco Qinyan plants are cylindrical, the height of the topping plant is 143.5cm, the stem circumference is 10.4cm, the pitch is 6.6cm, and the number of the collected leaves is 21-23; the leaves are oval, green, flat, uniform in stem and leaf angles, 67.3cm long in waist and 35.0cm wide; the field growth potential is strong, the transplanting time is about 60 days after the transplanting to bud emergence, and the field growth period is about 119 days. The Qin tobacco 96 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.
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 of morphology is high, so that the tobacco main cultivars are difficult to distinguish and identify. The detection method of the tobacco variety is mainly a field planting identification method, but 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 (small character index difference), difference (influenced by environment) among the same character year 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.
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, specific SNP molecular markers of specific main cultivars can be obtained by screening, and a tobacco variety identification method is designed according to the specific SNP markers.
Disclosure of Invention
The invention aims to provide a primer combination for identifying the Qin tobacco 96 of flue-cured tobacco.
Secondly, the invention also provides a flue-cured tobacco Qin tobacco 96 identification kit.
The invention further provides application of the primer combination or the kit in the aspect of identifying the 96 varieties of the flue-cured tobacco Qin tobacco.
Finally, the invention also provides a method for simply, quickly and efficiently identifying the flue-cured tobacco Qin 96.
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 cured tobacco Qin tobacco 96, the primer is designed aiming at 12 specific SNP locus flanking sequences of the cured tobacco Qin tobacco 96, and the primer is specifically designed according to different corresponding detection methods. The 12 SNP markers are specific SNP loci selected from flue-cured tobacco Qin tobacco 96 based on the whole-genome SNP typing results of main tobacco cultivars in China in recent years, the physical positions of the specific SNP loci are determined based on whole-genome sequence comparison of Hongda major pivot of cultivated tobacco cultivars, and the specific locus information is shown in the following table 1 and is respectively named as FP 01-FP 12. The gene sequence containing the 12 SNP loci is shown as SEQ ID NO 1-12.
TABLE 1 flue-cured tobacco, Qin tobacco, 96, 12 specific SNP site information
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, SNapshot method and MassARRAY molecular weight array technology to realize the variety identification of the flue-cured tobacco Qinyao 96.
96 identification kit for flue-cured tobacco and ash leafThe primer combination can also comprise PCR buffer solution and MgCl2dNTPs, PCR Enzyme, SAP buffer, iPLEX termination mix, iPLEX Enzyme, water, etc.
The primer combination or the kit is applied to the identification of the 96 varieties of flue-cured tobacco Qin tobacco. 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 Qin tobacco 96.
TABLE 2 genotypes of individual SNP sites in Qin tobacco 96 of flue-cured tobacco
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 Qin tobacco 96
The method for identifying the Qin tobacco 96 of the flue-cured tobacco 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
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 12 SNP sites in the detection sample are completely consistent with those of the flue-cured tobacco Qin tobacco 96, judging the tobacco sample to be the flue-cured tobacco Qin tobacco 96; the genotypes of 12 SNP loci FP 01-FP 12 in the flue-cured tobacco Qin tobacco 96 are AA, TT, CC, GG, TT, CC, AA, TT and AA in sequence.
The multiplex PCR amplification reaction in the step 1) comprises a reaction system of 0.5 mu L of 10 × PCR buffer solution and 25mM MgCl2mu.L of 0.4. mu.L, 0.1. mu.L of 25mM dNTPs, 0.2. mu.L of 5U/. mu.L PCR Enzyme, 1. mu.L of a mixture of 1. mu.M amplification primers (mixed in equal amounts), 1. mu.L of 10 ng/. mu.L tobacco sample genomic DNA, and water to 5. 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 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 XPLEX buffer solution, 0.2. mu.L of 10 XPLEX determination mix, 0.041. mu.L of 33U/. mu.L of iPLEX Enzyme and 0.94. mu.L of 1. mu.M mixture of extension primers (equal amount mixing) into the reaction product, and adding water 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 ℃.
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.
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 specific SNP markers suitable for identifying 96 cured tobacco Qin tobacco according to the inter-cultivar polymorphism SNP locus screening, the physical positions of the specific SNP markers are determined based on the whole genome sequence comparison of the cultivated tobacco cultivar safflower large gold, the specific locus information is shown in the table 1, and 12 gene sequences containing SNP loci are obtained by comparing the reference genome of the cultivated tobacco, as shown in SEQ ID NO: 1-12. Aiming at 12 specific SNP locus flanking sequences in the cured tobacco Qin tobacco 96, corresponding primers are designed according to different detection methods, and the method can be used for variety identification of the cured tobacco Qin tobacco 96. The invention preferably adopts a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology (MassARRAY molecular weight array platform), and two amplification primers and one extension primer are designed for each site based on the Assay Design Suit (Agena), and the primer combination is shown in the table 3.
In the method, in the identification of the flue-cured tobacco Qin tobacco 96 varieties, firstly, the genomic DNA of a sample to be detected is extracted, then SNP locus multiplex PCR amplification, SAP enzyme impurity removal and single base extension reaction are sequentially carried out according to the operation requirements of a MassARRAY system platform, a MALDI-TOF mass spectrometer is used for scanning a chip to obtain a typing result after mounting, and if the genotypes of SNPA markers FP 01-FP 12 in the detected sample are AA, TT, CC, GG, TT, CC, AA, TT and AA in sequence, the tobacco sample is judged to be the flue-cured tobacco Qin tobacco 96. The method has the advantages of simple operation, small sample consumption, short detection period, accurate identification result, good repeatability, high detection flux, rapidness, high efficiency and reliability, is a domestic and foreign first set of molecular detection system for the Qin tobacco 96 varieties of flue-cured tobaccos, provides a foundation and basis for the identification technical system for the Qin tobacco 96 of flue-cured tobaccos, and has good application and popularization prospects.
Detailed Description
The following examples are intended to illustrate the invention in further detail, but are not to be construed as limiting the invention in any way.
Example 1
The primer combination for identifying the flue-cured tobacco Qin tobacco 96 aims at the specific SNP locus marker design of the flue-cured tobacco Qin tobacco 96 and comprises the following steps:
1) whole genome SNP (single nucleotide polymorphism) typing detection of 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 DNA of the Tobacco sample is amplified by a whole genome, the product is randomly fragmented into fragments between 25 and 125bp, the fragments are re-suspended after being purified, the fragments are hybridized with 420K Tobacco SNP array, and each SNP is identified and connected through double-color connection reaction generated on the surface of a chip. And after the hybridization process is finished, carrying out rigorous washing to remove non-specific binding, after the connection reaction is finished, completing the steps of dyeing and washing the chip on a Gene Titan multi-channel automatic chip workstation, and finally scanning and outputting a result. And processing the data obtained by the chip analysis to obtain SNP typing results of different varieties.
2) Flue-cured tobacco Qinyan 96-specificity SNP locus screening
Selecting two types of site data, namely Poly high resolution and Mono high resolution, according to the data classification and recommendation type of a chip system, filtering to obtain a high-quality SNP typing result, reserving sites with 100% of Call rates in all detected varieties, further screening to remove sites with heterozygous typing in any variety, finally obtaining homozygous SNP sites in all detected varieties, and screening to obtain specific SNP sites in the Qin tobacco 96. Because a large number of repetitive sequences exist in tobacco, in order to avoid non-specific amplification in the design of detection primers, the 200bp sequences flanking the SNP locus are subjected to blast comparison with a reference genome, the locus without highly similar sequences in the genome is screened, a locus is selected on the chromosome with the polymorphic locus as a specific SNP marker of the Qin tobacco 96 by combining the distribution condition of the SNP locus on the chromosome, and the screening result is shown in the table 1.
3) Primer combination design for identifying 96 of flue-cured tobacco Qin tobacco
And carrying out chromosome positioning on the screened specific SNP loci in a reference genome to obtain upstream and downstream sequences containing the SNP loci, wherein the upstream and downstream sequences are shown as SEQ ID NO: 1-12. Two amplification primers and one extension primer were designed for each site based on the Assay Design Suit (Agena), and the primer combinations are shown in Table 3 above. The primers are all synthesized by Huada gene.
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, the MassARRAY has extremely high cost performance and is particularly suitable for large-scale typing detection of a limited number of SNP sites.
Example 2
The kit for identifying 96 of flue-cured tobacco Qin tobacco comprises 5mL of mixed solution (1 mu M) of the amplification primers in the embodiment 1, 5mL of 10 × PCR buffer solution and 25mM MgCl24mL, 25mM dNTPs 2mL, 5U/. mu.L PCR Enzyme 2mL, 1.7U/. mu.L SAP 3mL, 10 × SAP buffer 3mL, 10 × iPLEX buffer 2mL, 10 × iPLEX termination mix 2mL, 33U/. mu.L iPLEX Enzyme 1mL, 5mL of the mixture of extended primers in example 1 (1. mu.M), and 200mL of water.
Example 3
The method for identifying the Qin tobacco 96 of the flue-cured tobacco comprises the following steps:
1. DNA extraction of sample to be tested
Extracting a sample genome DNA by using a Gene Pure New way Plant Genomic DNA Kit (Gene Answer) by using a fresh leaf tissue of a tobacco sample; the DNA concentration was measured by a nucleic acid protein analyzer, NanoDrop ND-2000(Thermo Fisher Scientific), and the DNA was diluted to 10 ng/. mu.L for use.
2. MassARRAY assay
The operation is carried out according to the requirements of a MassARRAY system platform (Agena), and the reaction utilizes an iPLEX Gold reagent kit (Agena), which 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 PCR buffer (10 ×)0.5 mu L, MgCl20.4 μ L, dNTPs (25mM)0.1 μ L, PCREnzyme (5U/. mu.L) 0.2 μ L (25mM), 1 μ L of mixture of amplification primers (1 μ M), 1 μ L of test sample genomic DNA (10 ng/. mu.L), and water to make up to 5 μ 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 ℃.
2) SAP enzymatic reaction
Removing residual dNTPs and primers from the PCR product with SAP (shrimp alkaline phosphatase), adding SAP (1.7U/. mu.L) 0.3. mu. L, SAP buffer (10X) 0.17. mu.L to the PCR product of step 1), and making up to 7. mu.L with water; the reaction conditions are as follows: 40min at 37 ℃ and 5min at 85 ℃ to obtain a reaction product.
3) Single base extension reaction
Extension reaction was performed using iPLEX Reagent Kit, and 0.2. mu.L of iPLEX buffer (10X), 0.2. mu.L of iPLEX termination mix (10X), 0.041. mu.L of iPLEX Enzyme (33U/. mu.L), 0.94. mu.L of a mixture of extension primers (1. mu.M), and water were added to the reaction product of 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; and 3min at 72 ℃ to obtain an extension product.
4) Genotype detection
Adding 41 μ L of water and 15mg of clean resin (96-pore plate) into the extension product obtained in the step 3), shaking upside down for 15min for desalting, deionizing and interference-preventing treatment, centrifuging at 3200g for 5min, and taking the supernatant for later use; the supernatant was spotted onto 384-spot SpectroCHIP (chip) using a MassARRAY nanodispenseRS 1000 spotter; the chip was placed in a MassARRAY TypersWorkstation MA4, the chip was scanned using a MALDI-TOF (matrix assisted laser Desorption ionization time of flight) mass spectrometer, the scan was analyzed with Typer 4.0 software and the results were derived.
3. Comparison of the results
And (3) judging the obtained SNP marker detection result, wherein the detection result of 12 SNP loci in the detection sample is completely consistent with the result of the fingerprint spectrum of the Qinying 96, and judging that the sample is the flue-cured tobacco Qinying 96.
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 in example 3 is adopted to carry out detection and identification by taking 24 samples of known varieties as an example, the specificity of the method is verified, and the detection results are shown in the following table 4. Samples 1-24 in the table, which are involved in detection, are in turn: 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, Nanjiang No. 3, aromatic tobacco Yunxima No. 1, Yunxiang No. 2, Basma, Baibiao No. 1, Hubei tobacco No. 3, VAM, Burley-21, cigar Beinhart-1000, Havana-10 and Florida-301. Among samples participating in detection, flue-cured tobacco K326, safflower Dajinyuan, Zhongyan 100, Cuibi No. 1, Yunyan 85, Yunyan 87, Yunyan 97, Yunyan 100, Longjiang 911, Longjiang 981, Qin tobacco 96, Bina No. 1, Nanjiang No. 3, aromatic tobacco Yunxiangma No. 1, Yunxiang No. 2, Bailiangye No. 1 and Hubei tobacco No. 3 are all main cultivated varieties popularized and used in tobacco production in China in recent years.
Table 424 samples of known species
As can be seen from Table 4, the SNP site detection result of only the sample 12 in 24 samples is completely consistent with the fingerprint result of the Qin tobacco 96, which indicates that the detection method has specificity to the flue-cured tobacco Qin tobacco 96.
Sequence listing
SEQUENCE LISTING
<110> Zhengzhou tobacco institute of China tobacco general Co
<120> primer combination and kit for identifying 96 of flue-cured tobacco Qin tobacco, application and identification method
<170>PatentIn version 3.5
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP01 site (N is G or A)
<222>(1)..(201)
<400>1
TGATACTCAC CAACAGTATT CTGAAGACTC TTTAAACCCT GAAATTAACC CCCTTAATAT 60
CTCCTTAATA AACAATATTA GTTGTATGGA TGAAGATAAA NATCCACGAC ACCTGTCCCA 120
CAACAACTCA TTAGATGCAC AAATGGCCAA TGAAGTAAAA CACTTGCCTA GCACTCTCAA 180
TACCCCAAAT CCTATTAAAA T 201
<211>201
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<221> Gene sequence containing FP02 site (N is G or T)
<222>(1)..(201)
<400>2
GCCCCCAACA AGTAGTGACG CCATGTCCTT AGGCAGTGGA CTACGATCGT CATTTCCTTC 60
TCCTGGACAG TATAACGCCT CTTGTCGTTA TTCAACTTTC NACTCTTGAA TGCTATAGGA 120
TGACCCTCTT GCATCAGTAT GCCTTATATA GCAAAATCAG ACGCATTGGT CTGAACTTCA 180
AAAACTTTAG AGAAATCAGG C 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP03 site (N is A or C)
<222>(1)..(201)
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GCTCGAAAAT CGACGCCCTT ACTCCGGAAT TCGCCCGAGC TTGATATGTG GATACCCTTC 60
GATCGAGATG TCATTGTCTT GGTCAGTCTT TATGACTGAG NATCGATTTT GACTATACAC 120
AAACATAATA GTGTAAAAGA AGTGTCACGA CCCAAAATCT CACCTATCGT GATGACATCT 180
ATCTCAATAC TAGGCAAGCC G 201
<211>201
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<213> sequence
<221> Gene sequence containing FP04 site (N is A or G)
<222>(1)..(201)
<400>4
CGTTACCTCC TTCACTCTCA CTCATATATT CATGATCCTC ACCCTTACTT TCAGAAACCT 60
CCTCTTCTTC ACTCTGATTT TCTTCTTCTG TAGAATCATT NGCATGTTCA TCTGTCTTAT 120
CCTCTGTATC ACTCTCCTTC TCATCTCCAA ATACTCCCTC ATTCTCACTC TTTTCTTCTT 180
CATTGGGTAT GTTTTAAAAA T 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP05 site (N is C or T)
<222>(1)..(201)
<400>5
AATTGTGATT AGGTGGCATC CATTTTTGCC CCTTTATGTG CTGCTCCTAA GAGAGAGGCA 60
CTTAGAGCGA TGAGTGATGT AGAGTTGTCA CAGAGTATTT NCGGCATGGC TCTACGGGTG 120
TATGCCTCTC TTTTCTTTTT GTCGTTAAAT CTGTTTTACT ATTCTTAGCC CATTATTTTG 180
TTTTGACTTT GTTTTTCATA T 201
<211>201
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<213> sequence
<221> Gene sequence containing FP06 site (N is A or C)
<222>(1)..(201)
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GACAAAACCT CTTAATATGA CCCAAATCTC CACACTCGAA GCAACCTCTC CCAGCAAATG 60
GCGAGGGGGA CTGAAGGGAC CCCTGAGCTC CAGGATAACC NCTAGAAGAA CTGGGCGCAG 120
ATGTACCCTG AACTGATGGT GCCTGAGTCA AACTCTAAGC TAGAAGGGCT CTGAGAGATG 180
AGTGGCCCTA ATATGATCTG T 201
<211>201
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<213> sequence
<221> Gene sequence containing FP07 site (N is G or A)
<222>(1)..(201)
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CTTGCATCTA GGGTTCATCT CCTTCCTATT CTTGGCTATT CCAGTGTGAT TTCATGAGAA 60
ATCATTCTAT ATTTGTTATG ATCCTTGGGA TTTTAGACAG NCCTCTTCAT CTATATATGG 120
GTTCTTTCAA AACCTTAATT TCTTTTCTAT ACCTCCTAGA TCTGTACAGA TCTAGGACGA 180
TTTGAGTTTG TTTCTTGAAT G 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP08 site (N is C or T)
<222>(1)..(201)
<400>8
TCTTGGTATT CTCTGATAGA TCCGAAGAAC CCGAAGAGAA AGGGTCAGCC GACTACCACC 60
GTAGGCTAGT CTGATGAGCC TTCGGTGTTA GCTGCAGAGT NAGTAGATAT GCCCTCCACC 120
TCAGTAGATC CTTCTGCCAT TGCAGTTGCC ATGCCTCCAC CTTCAGCCGC AGTTCCTCCC 180
TCCTTGGCTT TGAAGCCGAT G 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP09 site (N is C or T)
<222>(1)..(201)
<400>9
CGATCCTGAA TCAATTTAAC CTTTTCCAAA GCATCCTGAA CTAAGTCTGT ACCCAATAGC 60
CTAGCCTCGC TCGGCTCGAG CCAACCACTA GAAACCGGCA NCGCCTACCT TACCAAGCCT 120
CATAGGTACC ATATGAATGC TCGACTGATA ATTGTTGTTG TAAGCAAACT CCGCAAGTGG 180
AAAGAACTGA TCCCAAGCAC C 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP10 site (N is C or T)
<222>(1)..(201)
<400>10
TGGCGGCTTA CATTCAATAT TTTCAAGCAC GTGATAATTG AAGGAAATAG AAATATACAA 60
TTGTCCAACA ACATACCCAT TATATTCTCA TACTTAAGAT NTGAAGAGAG TAGTGTGTAC 120
GCAGAACTTA CCCCTACCTA GTGAAGGTTG AGAGATTGTT TTCAATAATT TAAAGGGCAG 180
CCTGGTGCAC TAAACTCCCG C 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP11 site (N is C or T)
<222>(1)..(201)
<400>11
GTATACGTGG GGGCTGAGTA GTCTGTGGAG GTACACCTTT CCTGAGTCTG GGGAAATCCC 60
TCACCATATG ATGAGTGTCA CCACACTCAA AATAAGCCCT NGGAGGACGT GGATGCAGGA 120
ACTGGCTCGG AACTGATCGT TGAAAGCACC CCGTGCAGAA GGTGCATTAC ATAGTGGCAG 180
TGCATAATGA GGAACTTGAG G 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP12 site (N is G or A)
<222>(1)..(201)
<400>12
CCCTTCTCAA ACCCCATATG TGAAATTATA CTGAGTTTTT TTTATTGTTG TTGTTATTGT 60
GTTTATTTTC TAGGAATCTG ATACTGTTCT TACTATTACA NATATAGATT TAGTTGAAGT 120
AGTTAAGTAG TTATGATGTA GAGGGTATTT AAGTAAATAG TAGATTGGTT AGTTTAGGCA 180
GTTAAAATAG TATTAATACA C 201
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP01-F
<222>(1)..(30)
<400>13
ACGTTGGATG AAACCCTGAA ATTAACCCCC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP01-R
<222>(1)..(30)
<400>14
ACGTTGGATG ATGAGTTGTT GTGGGACAGG 30
<211>16
<212>DNA
<213> Artificial sequence
<221> primer FP01-E
<222>(1)..(16)
<400>15
GACAGGTGTC GTGGAT 16
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP02-F
<222>(1)..(30)
<400>16
ACGTTGGATG GGACAGTATA ACGCCTCTTG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP02-R
<222>(1)..(30)
<400>17
ACGTTGGATG CATACTGATG CAAGAGGGTC 30
<211>21
<212>DNA
<213> Artificial sequence
<221> primer FP02-E
<222>(1)..(21)
<400>18
TCTTGTCGTT ATTCAACTTT C 21
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP03-F
<222>(1)..(30)
<400>19
ACGTTGGATG TGTCATTGTC TTGGTCAGTC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP03-R
<222>(1)..(30)
<400>20
ACGTTGGATG AGGTGAGATT TTGGGTCGTG 30
<211>22
<212>DNA
<213> Artificial sequence
<221> primer FP03-E
<222>(1)..(22)
<400>21
CGGGTCAGTC TTTATGACTG AG 22
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP04-F
<222>(1)..(30)
<400>22
ACGTTGGATG ACCTCCTCTT CTTCACTCTG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP04-R
<222>(1)..(30)
<400>23
ACGTTGGATG GAAGGAGAGT GATACAGAGG 30
<211>19
<212>DNA
<213> Artificial sequence
<221> primer FP04-E
<222>(1)..(19)
<400>24
ATAAGACAGA TGAACATGC 19
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP05-F
<222>(1)..(30)
<400>25
ACGTTGGATG GAGCGATGAG TGATGTAGAG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP05-R
<222>(1)..(30)
<400>26
ACGTTGGATG GAAAAGAGAG GCATACACCC 30
<211>23
<212>DNA
<213> Artificial sequence
<221> primer FP05-E
<222>(1)..(23)
<400>27
TGTAGAGTTG TCACAGAGTA TTT 23
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP06-F
<222>(1)..(30)
<400>28
ACGTTGGATG AACCTCTCCC AGCAAATGGC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP06-R
<222>(1)..(30)
<400>29
ACGTTGGATG TCAGTTCAGG GTACATCTGC 30
<211>19
<212>DNA
<213> Artificial sequence
<221> primer FP06-E
<222>(1)..(19)
<400>30
ACTGAGCTCC AGGATAACC 19
<211>29
<212>DNA
<213> Artificial sequence
<221> primer FP07-F
<222>(1)..(29)
<400>31
ACGTTGGATG GTTATGATCC TTGGGATTT 29
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP07-R
<222>(1)..(30)
<400>32
ACGTTGGATG ATCTGTACAG ATCTAGGAGG 30
<211>21
<212>DNA
<213> Artificial sequence
<221> primer FP07-E
<222>(1)..(21)
<400>33
ACCCATATAT AGATGAAGAG G 21
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP08-F
<222>(1)..(30)
<400>34
ACGTTGGATG TGATGAGCCT TCGGTGTTAG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP08-R
<222>(1)..(30)
<400>35
ACGTTGGATG GCAATGGCAG AAGGATCTAC 30
<211>18
<212>DNA
<213> Artificial sequence
<221> primer FP08-E
<222>(1)..(18)
<400>36
CGGTGTTAGC TGCAGAGT 18
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP09-F
<222>(1)..(30)
<400>37
ACGTTGGATG TCTGTACCCA ATAGCCTAGC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP09-R
<222>(1)..(30)
<400>38
ACGTTGGATG ATGGTACCTA TGAGGCTTGG 30
<211>15
<212>DNA
<213> Artificial sequence
<221> primer FP09-E
<222>(1)..(15)
<400>39
CACTAGAAAC CGGCA 15
<211>31
<212>DNA
<213> Artificial sequence
<221> primer FP10-F
<222>(1)..(31)
<400>40
ACGTTGGATG CAATTGTCCA ACAACATACC C 31
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP10-R
<222>(1)..(30)
<400>41
ACGTTGGATG GGTAAGTTCT GCGTACACAC 30
<211>25
<212>DNA
<213> Artificial sequence
<221> primer FP10-E
<222>(1)..(25)
<400>42
CCCATTATAT TCTCATACTT AAGAT 25
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP11-F
<222>(1)..(30)
<400>43
ACGTTGGATG ACCATATGAT GAGTGTCACC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP11-R
<222>(1)..(30)
<400>44
ACGTTGGATG TTCAACGATC AGTTCCGAGC 30
<211>18
<212>DNA
<213> Artificial sequence
<221> primer FP11-E
<222>(1)..(18)
<400>45
ACACTCAAAA TAAGCCCT 18
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP12-F
<222>(1)..(30)
<400>46
ACGTTGGATG CTAGGAATCT GATACTGTTC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP12-R
<222>(1)..(30)
<400>47
ACGTTGGATG ACCCTCTACA TCATAACTAC 30
<211>23
<212>DNA
<213> Artificial sequence
<221> primer FP12-E
<222>(1)..(23)
<400>48
CTGATACTGT TCTTACTATT ACA 23
Claims (9)
1. A primer combination for appraising cured tobacco ash 96, its characterized in that: the primer is designed aiming at 12 specific SNP loci flanking sequences of 96 flue-cured tobacco Qin tobacco, the gene sequence containing the 12 SNP loci is shown as SEQ ID NO 1-12, and N of the gene sequence SEQ ID NO 1-12 is A, T, C, G, T, C, A, T, T, T, T, A 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. A flue-cured tobacco, ash-leaf tobacco 96 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 96 varieties of flue-cured tobacco Qin tobacco, and are characterized in that: the method comprises the following steps: taking the genomic DNA of the tobacco sample to carry out SNP typing detection, and if the genotypes of 12 SNP loci in the detected sample are completely consistent with those of the flue-cured tobacco Qin tobacco 96, judging the tobacco sample to be the flue-cured tobacco Qin tobacco 96; the genotypes of 12 SNP loci in the flue-cured tobacco Qin tobacco 96 are AA, TT, CC, GG, TT, CC, AA, TT and AA in sequence.
5. The method for identifying the cured tobacco Qin tobacco 96 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
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 12 SNP sites in the detection sample are completely consistent with those of the flue-cured tobacco Qin tobacco 96, judging the tobacco sample to be the flue-cured tobacco Qin tobacco 96; the genotypes of 12 SNP loci in the flue-cured tobacco Qin tobacco 96 are AA, TT, CC, GG, TT, CC, AA, TT and AA in sequence.
6. The method according to claim 5, wherein the multiplex PCR amplification reaction in step 1) comprises a reaction system of 0.5. mu.L of 10 × PCR buffer and 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: 30s at 94 ℃; 5 cycles of 94 ℃ for 5s, 52 ℃ for 5s, 80 ℃ for 5s, 40 cycles; 3min at 72 ℃.
7. The method of claim 5, 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.
8. The method of claim 5, 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 ℃.
9. The method of claim 5, 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 onto a chip, and scanning the chip by using a MALDI-TOF mass spectrometer to obtain an SNP genotype detection result.
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| CN201710557210.4A CN107345252B (en) | 2017-07-10 | 2017-07-10 | Primer combination and kit for identifying 96 of flue-cured tobacco Qin tobacco, application and identification method |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101065481A (en) * | 2004-04-29 | 2007-10-31 | 美国无烟烟草公司 | Nicotiana nucleic acid molecules and uses thereof |
| CN102757956A (en) * | 2012-07-18 | 2012-10-31 | 云南省烟草农业科学研究院 | Tobacco genome molecular marker probe and sequence collective group as well as acquiring method and application thereof |
| WO2013080045A2 (en) * | 2011-11-28 | 2013-06-06 | Anglo Netherlands Grain B.V. | Method for differentiating fertile and sterile plant lines by detection of polymorphic markers in chloroplast dna |
| CN104726564A (en) * | 2015-02-15 | 2015-06-24 | 西南大学 | Primer set, kit and method for identifying different tobacco varieties |
-
2017
- 2017-07-10 CN CN201710557210.4A patent/CN107345252B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101065481A (en) * | 2004-04-29 | 2007-10-31 | 美国无烟烟草公司 | Nicotiana nucleic acid molecules and uses thereof |
| WO2013080045A2 (en) * | 2011-11-28 | 2013-06-06 | Anglo Netherlands Grain B.V. | Method for differentiating fertile and sterile plant lines by detection of polymorphic markers in chloroplast dna |
| CN102757956A (en) * | 2012-07-18 | 2012-10-31 | 云南省烟草农业科学研究院 | Tobacco genome molecular marker probe and sequence collective group as well as acquiring method and application thereof |
| CN104726564A (en) * | 2015-02-15 | 2015-06-24 | 西南大学 | Primer set, kit and method for identifying different tobacco varieties |
Non-Patent Citations (5)
| Title |
|---|
| Construction of a high-density SNP genetic map in fluecured tobacco based on SLAF-seq;Gong D et al.;《Mol Breeding》;20160731;第36卷(第7期);摘要，材料和方法，结果，讨论，图3，表1 * |
| SNP-based genetic linkage map of tobacco (Nicotiana tabacum L.) using next-generation RAD sequencing;Xiao B et al.;《J of Biol Res-Thessaloniki》;20151006;第22卷;摘要，结果，方法 * |
| 一种通过寡核苷酸多态性芯片识别基因型的方法;符碧玲;《中国优秀硕士学位论文全文数据库 基础科学辑》;20110315(第3期);第A006-95页 * |
| 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;第40卷(第3期);第397-404页 * |
| 植物SNP在线工具的开发及其在烟草中的应用;周东洁;《中国优秀硕士学位论文全文数据库 农业科技辑》;20170315(第3期);第D047-453页 * |
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