CN107354202B - Primer combination and kit for identifying flue-cured tobacco K326, application and identification method - Google Patents
Primer combination and kit for identifying flue-cured tobacco K326, application and identification method Download PDFInfo
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Abstract
The invention discloses a primer combination and a kit for identifying flue-cured tobacco K326, and an application and an identification method thereof, belonging to the technical field of biomolecule identification. The tobacco 420K high-density SNP chip is used for carrying out whole genome SNP typing on main tobacco cultivars in China in recent years, 10 specific SNP markers suitable for identifying flue-cured tobacco K326 are obtained by screening according to polymorphism SNP sites among varieties, the physical positions of the specific SNP markers are determined based on whole genome sequence comparison of safflower large gold of cultivated tobacco varieties, and the sequences containing the SNP sites are shown as SEQ ID NO: 1-10. Aiming at the SNP locus flanking sequence of the flue-cured tobacco K326, corresponding primers are designed according to different detection methods, and the method can be used for variety identification of the flue-cured tobacco K326. The flue-cured tobacco K326 identification method is established based on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, and is short in detection period, high in flux and accurate in result.
Description
Technical Field
The invention relates to a primer combination for identifying flue-cured tobacco K326, 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 flue-cured tobacco variety K326 is obtained by hybridization breeding of the Northup King Seed Company (Northup King Seed Company), Yunnan province is introduced from the United states in 1985, the crop variety approval committee in Yunnan province is approved as a promoted variety in 1988, the national flue-cured tobacco variety regional test is participated in 1986-1988, and the national tobacco variety approval committee in 1989 is approved as a national promoted variety. Flue-cured tobacco K326 is cylindrical or tower-shaped, the height of the flue-cured tobacco K326 is 110-130 cm, the pitch is 4-4.89 cm, the stem circumference is 7-8.90 cm, the number of leaves is 24-26, and 18-21 leaves can be picked; the waist leaf is oblong in shape, green in leaf color, gradually sharp in leaf tip, wavy in leaf edge, wrinkled in leaf surface, small in leaf ear, thin in main vein, medium in leaf thickness, delicate in mesophyll tissue and large in stem and leaf angle; inflorescence is concentrated, and corolla is light red. The flue-cured tobacco K326 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 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.
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 flue-cured tobacco K326.
Secondly, the invention also provides a flue-cured tobacco K326 identification kit.
The invention further provides application of the primer combination or the kit in flue-cured tobacco K326 variety identification.
Finally, the invention also provides a method for simply, quickly and efficiently identifying the flue-cured tobacco K326.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the primer combination is used for identifying flue-cured tobacco K326, the primer is designed aiming at 10 specific SNP locus flanking sequences of the flue-cured tobacco K326, and the primer is specifically designed according to different corresponding detection methods. The 10 SNP markers are specific SNP loci 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 loci are determined based on the whole genome sequence comparison of Honghuadajinyuan of cultivated tobacco cultivars, and the specific locus information is shown in the following table 1 and is respectively named as FP 01-FP 10. The gene sequence containing the 10 SNP loci is shown as SEQ ID NO 1-10.
TABLE 1 flue-cured tobacco K326 10 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, SNap shot method and MassARRAY molecular weight array technology to realize variety identification of flue-cured tobacco K326.
The flue-cured tobacco K326 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 flue-cured tobacco K326 varieties. 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 10 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 K326.
TABLE 2 genotypes of various SNP sites in flue-cured tobacco K326
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 K326
The flue-cured tobacco K326 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
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 10 SNP sites in a detection sample are completely consistent with that of flue-cured tobacco K326, judging that the tobacco sample is flue-cured tobacco K326; the genotypes of 10 SNP sites FP 01-FP 10 in the flue-cured tobacco K326 are TT, AA, CC, AA, TT and CC 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: 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 mixture (equal amount mixing) 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.
The invention has the beneficial effects that:
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 10 specific SNP markers suitable for identifying flue-cured tobacco K326 by screening according to polymorphism SNP sites among varieties, the physical positions of the specific SNP markers are determined based on whole genome sequence comparison of big gold dollars of safflower of cultivated tobacco varieties, the specific site information is shown in the table 1, and 10 gene sequences containing SNP sites are obtained by comparing reference genomes of cultivated tobacco, as shown in SEQ ID NO: 1-10. Corresponding primers are designed according to different detection methods aiming at 10 specific SNP site flanking sequences in the flue-cured tobacco K326, and the method can be used for variety identification of the flue-cured tobacco K326. 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 flue-cured tobacco K326 variety identification, firstly extracting genome DNA of a sample to be detected, then sequentially carrying out SNP locus multiplex PCR amplification, SAP enzyme impurity removal and single base extension reaction according to the operation requirement of a MassARRAY system platform, carrying out mounting, and then scanning a chip by using a MALDI-TOF mass spectrometer to obtain a typing result, wherein if the genotypes of SNPA markers FP 01-FP 10 in the detected sample are TT, AA, CC, AA, TT and CC in sequence, the tobacco sample is judged to be flue-cured tobacco K326. 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 flue-cured tobacco K326 variety molecular detection system, provides basis and basis for a flue-cured tobacco K326 identification technology system, 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 K326 is designed aiming at the specific SNP locus marker of the flue-cured tobacco K326, 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 K326 specific SNP site 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 flue-cured tobacco K326. 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, one locus is selected on the chromosome with the polymorphic locus in combination with the distribution condition of the SNP locus on the chromosome, two loci with better chromosome polymorphism are selected as specific SNP markers of flue-cured tobacco K326, and the screening result is shown in the table 1.
3) Primer combination design for identifying flue-cured tobacco K326
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: 1-10. 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 flue-cured tobacco K326 identification kit comprises 5mL of a 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 flue-cured tobacco K326 identification method 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 muL、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 10 SNP sites in the detection sample is completely consistent with the K326 fingerprint result, and judging that the sample is flue-cured tobacco K326.
The MassARRAY molecular weight array platform based on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology can design 10-fold PCR reaction and genotype detection aiming at 10 SNP sites, and has flexible experimental design and high accuracy of typing results. 396 samples can be simultaneously detected for 10 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 sample 1 in 24 samples is completely consistent with the fingerprint result of flue-cured tobacco K326, which indicates that the detection method has specificity to flue-cured tobacco K326.
Sequence listing
SEQUENCE LISTING
<110> Zhengzhou tobacco institute of China tobacco general Co
<120> primer combination and kit for identifying flue-cured tobacco K326, application and identification method
<170>PatentIn version 3.5
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP01 site (N is C or T)
<222>(1)..(201)
<400>1
GGTTGTTTCA TCAAGTCCAG AAAGGCGGAC CACACTTCTT CGTTGAGCGA CCGCAGAAAA 60
GTAATGCTGA CCGCAGAAGC TCTTGTACAA CCGTAGATTT NCTCTGCGGA CCACACTTAC 120
AGGAGCCCAT CTTAGGTGCT AAATGGAAAA CCCTATGAAA TTTTCTCCCA ACAGTGCGGA 180
GTAATAGTGC GGACTTTCAC A 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP02 site (N is C or T)
<222>(1)..(201)
<400>2
CATTCACAAT TCACCATACA TCTATCAAGT CTTTCTAGAA TTCTTTGGTT GAGCGATCAT 60
TTATTCGTCC AGGTGTATTT ACTGCCTCTA TAACCAATAT NAATTAATTG ACAATAGTTT 120
AGGCATCGTA TGAAGTTGTC ACATTTTGTG TTACTAACAG CATTTCCCCC AAACTTTTCA 180
GAGGCCTATA TGATTTCATT A 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP03 site (N is G or A)
<222>(1)..(201)
<400>3
TGTCGGAATG GAATTTTGGA ATCGGTGTCC TTCTTCGCAA TCGCGTGATG GGGCTTACGA 60
TCGCGTAGAT CATTCATGGA GGCAGTCATGTTTGTAATTC NCGTTCGCAT ATTAAGTGTT 120
GTGATTGTGT TGGTTGATCA GGTGAAGCAT TGCGAATGCG TGGGTTAAGT GGAGTCTGCG 180
TAAGGTTAAG TGGACCAAAG T 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP04 site (N is G or A)
<222>(1)..(201)
<400>4
CTGATCAGAG GTTGCCCAAT AGAGGCCTGC CCATCGATTA TAGCTCAATG ATAATGAAAA 60
TACTTTTAAT ACTGTATAAA TGTAAACTCT TTGATCTTTT NGCCGGAAGA AGGAAATACT 120
CAACTGAATA TGCAGTCCCA ATAAGAGGAA TATGGTAACT TACAAAACTA GGAAAATGTA 180
TGTAATTTGC GAGACTAGCA A 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP05 site (N is T or C)
<222>(1)..(201)
<400>5
GTCCATTCAG TTACTAGTTG GGATCTAGAT GTGCTTTCTT CGGGGAACAC AAGATGGAGA 60
ACTTATCCCG GCCAAAACAA ATAGATCTAG GTCCACACCC NGATTCATCA TCATCACTGT 120
CAATGTCACT TGCATTCATC CTATTCTGCT GACCTCTTCG CCGTACAAGG AAGACATTAA 180
AATAGTAGCT TACCAAAGAT T 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP06 site (N is T or C)
<222>(1)..(201)
<400>6
CCAAACATGC CCTTAAGGTG TAGTTAGGTA TCCTTCAATC GTTTTTGTTC ATTATTCTTT 60
AATCTTGCCT CATATTCTTG CGTTGCCTTC AATATAGTGA NCAGGGACAA AAAAGTTCAC 120
TATGTGTATA CAACTTAAAT TATTTTTTCT TTTTTCTTTT TTTCACTTTT TTTCTATTTT 180
ATATTTAGTT AAAGGGATCT T 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP07 site (N is G or A)
<222>(1)..(201)
<400>7
ATTGTTAGCG AACCTAATTT TGGACTTGAG AAATAGCTGT AGAGTGTTGG ACTGGTGTGA 60
TAGCACCTGC GAGGGTCATG GTCACAGGTG CAAGGCCGCA NGTGCGGGGA TGGCATTGTA 120
TAAGCGAGTT TGACTGGGTT GCTGATGTGT CGCATGTGCG AGGGAAATGC CGAATCTATG 180
AGCCCGCAGA TACGTGGCTC C 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP08 site (N is C or T)
<222>(1)..(201)
<400>8
GGGGGACAAG GAGTGGGGAA CAATGGAGGA AAAAAATTGA GTGGAGACAC ACCTGGAAAG 60
CGGGAAATAT TCAAGCACGA TCAAAAATTA GGTGCTCACA NATCTAAATA TCCAAAGTCT 120
TCACCATATG GCACTTCGTG AAGTTTTATT TTTTAAGCGG GTCGGGAGAT AGATTTTTTC 180
GTTTTTTTAA ATGGATATCA T 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP09 site (N is C or T)
<222>(1)..(201)
<400>9
AAACCCCTGC TCAAATATCC ATTTTATCAC TGCTGCAAAA CTCTGAGGTG CATAGGAATG 60
CCTTGATGAA GGTATTGAAT GAAGTTTATG AACCCAATAA NATCACCAGT GGAGAGATGG 120
CCAACATGGT AGGGCAAGTG TTGGAAAGCC ACAAGATCAC TTTTCATGAA AGACGAGCTA 180
TCACCAGAAG GACTAAGTCA C 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP10 site (N is T or C)
<222>(1)..(201)
<400>10
TATCAACTCC AATTACGGAT GCATCATTGT GCTTTATTCC AAGTGGATTA CCAATAGTAT 60
TCCATATGAA TTTAGCCGCA TCACTTCCCA TAAAGATATG NTGTATAGTT TCACGTTGTG 120
GATGCCTGCA ATAAGAACAT CTTAAAACAA TGTTAGTACC AAACCTGTCC ATAGTATCAT 180
CAAAAAGGTA ATCTTTTTTG T 201
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP01-F
<222>(1)..(30)
<400>11
ACGTTGGATG AGTAATGCTG ACCGCAGAAG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP01-R
<222>(1)..(30)
<400>12
ACGTTGGATG TTAGCACCTA AGATGGGCTC 30
<211>20
<212>DNA
<213> Artificial sequence
<221> primer FP01-E
<222>(1)..(20)
<400>13
TCTTGTACAA CCGTAGATTT 20
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP02-F
<222>(1)..(30)
<400>14
ACGTTGGATG CGTCCAGGTG TATTTACTGC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP02-R
<222>(1)..(30)
<400>15
ACGTTGGATG GTGACAACTT CATACGATGC 30
<211>22
<212>DNA
<213> Artificial sequence
<221> primer FP02-E
<222>(1)..(22)
<400>16
TTACTGCCTC TATAACCAAT AT 22
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP03-F
<222>(1)..(30)
<400>17
ACGTTGGATG GATCATTCAT GGAGGCAGTC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP03-R
<222>(1)..(30)
<400>18
ACGTTGGATG GCTTCACCTG ATCAACCAAC 30
<211>18
<212>DNA
<213> Artificial sequence
<221> primer FP03-E
<222>(1)..(18)
<400>19
ACACTTAATA TGCGAACG 18
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP04-F
<222>(1)..(30)
<400>20
ACGTTGGATG CTGCCCATCG ATTATAGCTC 30
<211>29
<212>DNA
<213> Artificial sequence
<221> primer FP04-R
<222>(1)..(29)
<400>21
ACGTTGGATG CAGTTGAGTA TTTCCTTCT 29
<211>17
<212>DNA
<213> Artificial sequence
<221> primer FP04-E
<222>(1)..(17)
<400>22
TATTTCCTTC TTCCGGC 17
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP05-F
<222>(1)..(30)
<400>23
ACGTTGGATG GAACTTATCC CGGCCAAAAC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP05-R
<222>(1)..(30)
<400>24
ACGTTGGATG GAGGTCAGCA GAATAGGATG 30
<211>17
<212>DNA
<213> Artificial sequence
<221> primer FP05-E
<222>(1)..(17)
<400>25
GATCTAGGTC CACACCC 17
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP06-F
<222>(1)..(30)
<400>26
ACGTTGGATG CCTCATATTC TTGCGTTGCC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP06-R
<222>(1)..(30)
<400>27
ACGTTGGATG GTTGTATACA CATAGTGAAC 30
<211>20
<212>DNA
<213> Artificial sequence
<221> primer FP06-E
<222>(1)..(20)
<400>28
AGTGAACTTT TTTGTCCCTG 20
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP07-F
<222>(1)..(30)
<400>29
ACGTTGGATG TGTTGGACTG GTGTGATAGC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP07-R
<222>(1)..(30)
<400>30
ACGTTGGATG TCGCTTATAC AATGCCATCC 30
<211>15
<212>DNA
<213> Artificial sequence
<221> primer FP07-E
<222>(1)..(15)
<400>31
ATGCCATCCC CGCAC 15
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP08-F
<222>(1)..(30)
<400>32
ACGTTGGATG AGCGGGAAAT ATTCAAGCAC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP08-R
<222>(1)..(30)
<400>33
ACGTTGGATG CGAAGTGCCA TATGGTGAAG 30
<211>22
<212>DNA
<213> Artificial sequence
<221> primer FP08-E
<222>(1)..(22)
<400>34
TGAAGACTTT GGATATTTAG AT 22
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP09-F
<222>(1)..(30)
<400>35
ACGTTGGATG AGGTGCATAG GAATGCCTTG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP09-R
<222>(1)..(30)
<400>36
ACGTTGGATG TACCATGTTG GCCATCTCTC 30
<211>21
<212>DNA
<213> Artificial sequence
<221> primer FP09-E
<222>(1)..(21)
<400>37
GGGGATCTCT CCACTGGTGA T 21
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP10-F
<222>(1)..(30)
<400>38
ACGTTGGATG ATTTAGCCGC ATCACTTCCC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP10-R
<222>(1)..(30)
<400>39
ACGTTGGATG TATTGCAGGC ATCCACAACG 30
<211>23
<212>DNA
<213> Artificial sequence
<221> primer FP10-E
<222>(1)..(23)
<400>40
GCTCCACAAC GTGAAACTAT ACA 23
Claims (9)
1. A primer combination for appraising flue-cured tobacco K326, its characterized in that: the primer is designed aiming at 10 specific SNP locus flanking sequences of flue-cured tobacco K326, the gene sequence containing the 10 SNP loci is shown as SEQ ID NO 1-10, and N of the gene sequence SEQ ID NO 1-10 is T, T, A, A, C, C, A, T, T, C in sequence.
2. The primer combination of claim 1, wherein: the primer sequences of the 10 specific SNP sites are shown as SEQ ID NO. 11-40.
3. Flue-cured tobacco K326 identification kit comprising a primer combination according to claim 1 or 2.
4. The primer combination of claim 1 or 2 and the kit of claim 3 are used for identifying the variety of flue-cured tobacco K326, 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 10 SNP loci in the detected sample are completely consistent with that of the flue-cured tobacco K326, judging that the tobacco sample is the flue-cured tobacco K326; the genotypes of 10 SNP loci in the flue-cured tobacco K326 are TT, AA, CC, AA, TT and CC in sequence.
5. The method for identifying flue-cured tobacco K326 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 10 SNP sites in a detection sample are completely consistent with that of flue-cured tobacco K326, judging that the tobacco sample is flue-cured tobacco K326; the genotypes of 10 SNP loci in the flue-cured tobacco K326 are TT, AA, CC, AA, TT and CC 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: 2min at 95 ℃; 45 cycles of 95 ℃ for 30s, 56 ℃ for 30s, and 72 ℃ for 1 min; 5min 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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| CN109912543B (en) * | 2019-03-11 | 2020-11-24 | 云南农业大学 | Pasima A and preparation method thereof |
| CN111349713B (en) * | 2020-03-19 | 2022-10-04 | 中国烟草总公司海南省公司 | Set of KASP primer groups for detecting cigar resources and application thereof |
| CN112575112A (en) * | 2020-12-25 | 2021-03-30 | 红塔烟草(集团)有限责任公司 | Specific sequence for identifying K326 roasted variety, kit and use method thereof |
| CN114507747B (en) * | 2021-09-16 | 2022-11-15 | 贵州省烟草科学研究院 | Tobacco SNP marker developed based on whole genome re-sequencing and KASP technology and application thereof |
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