CN107345251B - Primer combination and kit for identifying flue-cured tobacco Longjiang 911, application and identification method - Google Patents
Primer combination and kit for identifying flue-cured tobacco Longjiang 911, 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 Longjiang 911, an application and an identification method, and belongs 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, 16 specific SNP markers suitable for identifying cured tobacco Longjiang 911 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 big gold dollars of safflower of cultivated tobacco varieties, and the gene sequences containing the SNP sites are shown as SEQ ID NO: 1-16. Corresponding primers are designed according to different detection methods aiming at the SNP locus flanking sequence of the flue-cured tobacco Longjiang 911, and can be used for variety identification of the Longjiang 911. The Longjiang 911 identification method established based on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology has the advantages of short detection period and high flux.
Description
Technical Field
The invention relates to a primer combination for identifying cured tobacco Longjiang 911, 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 Longjiang 911 is obtained by hybridization and pedigree breeding by using Longjiang 851 (winnel) as a female parent and CV91 as a male parent, which are obtained from the tobacco scientific research of Heilongjiang province, and is approved by the national tobacco variety approval committee in 12 months in 2000. The plant type of Longjiang 911 is tower-shaped, the natural plant height is 193.3cm, the pitch is 5.98cm, the stem circumference is 8.62cm, and the number of effective leaves is 15.8. The waist leaf is 78.2cm long, the width is 34.0cm, the top leaf is 54.7cm long, the width is 22.8cm, the waist leaf is oblong, the leaf tip is sharp, the leaf color is green, the leaf surface is wrinkled, the leaf margin is wavy, the leaf and the ear are medium, the mesophyll tissue is fine, and the angle of the stem and the leaf is medium; the field growth is regular, and the growth potential is strong; inflorescence is concentrated, and corolla is light red; transplanting until the bud grows for 44 days, transplanting until the central flower blooms for 49 days, the field growth period is about 120 days, and the full growth period is 178 days. Longjiang 911 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 cured tobacco Longjiang 911.
Secondly, the invention also provides a flue-cured tobacco Longjiang 911 identification kit.
The invention further provides application of the primer combination or the kit in identification of varieties of flue-cured tobacco Longjiang 911.
Finally, the invention also provides a method for simply, quickly and efficiently identifying the flue-cured tobacco Longjiang 911.
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 Longjiang 911, the primer is designed aiming at 16 specific SNP locus flanking sequences of the cured tobacco Longjiang 911, and the primer is specifically designed according to different corresponding detection methods. The 16 SNP markers are specific SNP sites selected from flue-cured tobacco Longjiang 911 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 FP 01-FP 16. The gene sequence containing the 16 SNP loci is shown as SEQ ID NO 1-16.
TABLE 1 flue-cured tobacco Longjiang 911 information of 16 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 the variety identification of the flue-cured tobacco Longjiang 911.
The flue-cured tobacco Longjiang 911 identification kit can 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 Longjiang 911 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 the 16 SNP sites in the detected sample are completely consistent with the results shown in the following table 2, judging that the tobacco sample is the cured tobacco Longjiang 911.
TABLE 2 genotypes of the individual SNP sites in flue-cured tobacco Longjiang 911
The invention preferably adopts a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology (MassARRAY molecular weight array platform), and designs two amplification primers and one extension primer aiming at each site based on the Assay Design Suit (Agena), wherein the primer combination is shown in the following table 3.
TABLE 3 primer combinations designed based on the specific SNP site of flue-cured tobacco Longjiang 911
The method for identifying the cured tobacco Longjiang 911 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 16 SNP sites in a detection sample are completely consistent with that of the cured tobacco Longjiang 911, judging that the tobacco sample is the cured tobacco Longjiang 911; the genotypes of 16 SNP sites FP 01-FP 16 in flue-cured tobacco Longjiang 911 are AA, TT, CC, TT, AA, TT, GG, AA, CC, AA, GG 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: 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 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, 16 specific SNP markers suitable for identifying cured tobacco Longjiang 911 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 Honghuadajinyuan of cultivated tobacco varieties, the specific site information is shown in the table 1, and 16 gene sequences containing SNP sites are obtained by comparing and cultivating reference genomes of tobacco, as shown in SEQ ID NO: 1-16. Corresponding primers are designed according to different detection methods aiming at 16 specific SNP site flanking sequences in the flue-cured tobacco Longjiang 911, and can be used for variety identification of the flue-cured tobacco Longjiang 911. 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 Longjiang 911 variety identification, firstly extracting the 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 16 in the detected sample are AA, TT, CC, TT, AA, TT, GG, AA, CC, AA, GG and AA in sequence, the tobacco sample is judged to be the flue-cured tobacco 911. 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 Longjiang 911 variety molecular detection system, provides a foundation and a basis for a flue-cured tobacco Longjiang 911 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
A primer combination for identifying flue-cured tobacco Longjiang 911 is designed aiming at a specific SNP locus marker of the flue-cured tobacco Longjiang 911, 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 Longjiang 911 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 the cured tobacco Longjiang 911. 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 sites are subjected to blast comparison with a reference genome, sites without highly similar sequences in the genome are screened, one site is selected on the chromosome with polymorphic sites as a specific SNP marker of the flue-cured Longjiang 911 in combination with the distribution condition of the SNP sites on the chromosome, and the screening result is shown in the table 1.
3) Primer combination design for identifying flue-cured tobacco Longjiang 911
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: 1-16. 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 Longjiang 911 identification kit 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 SAP3mL, 10 × SAP buffer 3mL, 10 × iPLEX buffer 2mL, 10 × iPLEX termination mix 2mL, 33U/. mu.LiPLEX 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 cured tobacco Longjiang 911 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: 2min at 95 ℃; 45 cycles of 95 ℃ for 30s, 56 ℃ for 30s, and 72 ℃ for 1 min; 5min 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 16 SNP loci in the detection sample is completely consistent with the fingerprint result of Longjiang 911, and judging that the sample is flue-cured Longjiang 911.
The MassARRAY molecular weight array platform based on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology can design 16-fold PCR reaction and genotype detection aiming at 16 SNP sites, and has flexible experimental design and high accuracy of typing results. 396 samples can be simultaneously detected for 16 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 result of the SNP site detection of only sample 10 in 24 samples is completely consistent with the fingerprint result of Longjiang 911, which indicates that the detection method has specificity to the flue-cured tobacco Longjiang 911.
Sequence listing
SEQUENCE LISTING
<110> Zhengzhou tobacco institute of China tobacco general Co
<120> primer combination and kit for identifying flue-cured tobacco Longjiang 911, 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 A)
<222>(1)..(201)
<400>1
CGTTTTTGTC CCAATTGGGT TTTTCGGCAA GGTTTTAGTA AGGCAACAGT GGATCGTGTT 60
AACTTATAAT TGAAGGTCGT TCACGAATCG GTATTAAAAA NTATGTTTAT AGTATCTGAG 120
ATTTCTTTAC AATCAACCTC GAATACTAGG GACCCTCAGA TATTGAATTG TTTTAGCAAG 180
GAAATTATTT CGAAATGAAA G 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP02 site (N is C or T)
<222>(1)..(201)
<400>2
TTAGATCTGA GATTCGAGGC GATGGAGGGG GATTTGAGAG GATTTGTTTG GAGATTTGGG 60
ATGAGGAGGT GGAGGGGAGT GTGAGGTGTT AATTTCAGGG NGGTTGGTGG TAGTGCCGCC 120
ACCAGTAGCG GTGGGATTTC AAGGGCGGCT ACTAGGGTTC CGGTCTGGTT GGAGAGGGTG 180
AGATGAGAGA CGAGACGGAG G 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP03 site (N is C or T)
<222>(1)..(201)
<400>3
CGGGAGGTAG AGATGAGAAC TTTTTATCAG CTAGTAGTGG AGATAGCTCG GAGGATTGAG 60
GGTTACCGTC AGAGAGGTAG AAAACAGATG CAGAGGGACA NGGGGGTCCG TTATTCTGGG 120
GAGTTTAGAG GTGCCCCGGC AGGGGGTTGA GGCCAGTTTG GGAGGGGTCA GCCTAGCAGG 180
CCCACATATC CAGCACCACC G 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP04 site (N is T or C)
<222>(1)..(201)
<400>4
GAATAGACGC CCTCTGCCAT GCTGAAATTG ACCCCCATGA GGAGCACCCC CAAAACTACC 60
AGATCCACGA GGCCTCTTGG CCTCCCTCTC CACTCGCTCC NGATGGTGAA TCAACTCTAT 120
GTCCCTAGCA ATGTTAACAA CCTCCTCAAA AGAAGCACCA GATACCCTCT CTCTAGTCGT 180
AAGAATCCGT AACTAATACA A 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP05 site (N is G or T)
<222>(1)..(201)
<400>5
ACCCTGTCTT CACCAGCTGG GATATGCAAC AACCTCCACC TTCTCCTGCC CAACAAGAAA 60
ATCCACCCAC TCAAGCAGAA GAACAAGCTC AAACACAGGA NAACATGCAA CAACAAGAAC 120
AAAATCTAGA GGAGGAAGAA CACATCCATC ACCAGGGGGA ACCAAGGGTG GCATTCTCAA 180
ACCTAAACGA GGTAAAAATT C 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP06 site (N is C or A)
<222>(1)..(201)
<400>6
GGTCACATAG ACGATGTCGA ACTCACTTAG CAGTACTTTC CACTTCGCCA ACTTGCCAGT 60
AGGCATAGGT GTCTGAAAAA TATACTTTAA AGGGTCCTTT NTCAATATGA GGTATGTGGT 120
GTATGCATAG AAATAATGCC TCAGCCTCTG GGCTTATCCA TGTCAGGGAA CATCAAGTAC 180
GCTCAAAAAA AGAATACCGA G 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP07 site (N is G or T)
<222>(1)..(201)
<400>7
CTTTTGAGTA ACTCCTTGAT GTACTTATGT TGGTGTATCA TGGTTCCCTT TTCTGTTTGC 60
TTGACTTGAA GCCCTAGGAA GAACTTAATT CTCCCATTAT NCTCATTTTA AATTCACTTC 120
CCATTAGTTC AGCAAATGCA TTGCAAAGAT ATTCGTTCGT GGCTCCAAAA ATGATGCCAT 180
CCACATAAAT TTGCACTTTC A 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP08 site (N is C or T)
<222>(1)..(201)
<400>8
GGAAGAAAGG AAAAAAATGA CTTATTGGAG TGAGGCTGCT TGCATCAATG GTCATGACAT 60
GCACTTTGGA TTAATAAGCA TAGTCCATTC AGTCAATCCT NTTTCACTCT TCACCCTTTT 120
ATGCCTGAGG TTTCCATAAT CTGGTTCCTT TGCAAAGTCA ACAACCTTAT TCGGCTTGCG 180
GTGCCCTGAA AGGTTTTCAC C 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP09 site (N is G or A)
<222>(1)..(201)
<400>9
ACTCTGAATT TCTAATAAAA TAATCTTGTT TTGATTGTCA CTTAAAATTG GAAAAAACTC 60
TCTATATACT CCCTTCCGGG GGTGTAGGTA AAAAGGAGGT NTGGCAAAGA GCATATATGT 120
GATGACGGTA AACTCGTGGT CGTGTACGGT AAAGATGTGG GCAATTTAGC CAGATGATAG 180
TATGTGCTAT GATTCAGTCA T 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP10 site (N is G or T)
<222>(1)..(201)
<400>10
TATCACCACC TATACAACAG CAGTGAAGGG AACTGATTTA GCTTCCCATG GGATTGAATC 60
AGTTTTGCTA AAGAAATTTG AGGAGACTCA TACGAGGGGA NCCCTGATGT GGTACTCGCT 120
TTTGCTGAGC ATTCCATAGA CTCCTTTGAG ATGTTCGCAG ACTCTTTCAT CAAAGCCCAT 180
GCCGGGGCCA GAAAGGTCCA G 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP11 site (N is T or G)
<222>(1)..(201)
<400>11
TGGCTTTAGC ATAGTTATCG TTATCACCTT TTCGGCCAAC GTAACCATAT CTAGCAGCTG 60
AGGATTGTAA CCAATGTAGA ACATCAAAAT TCTTGTATGA NAAAGCTGTA ACTTTAGGAA 120
ACCCTCAGAC ATGTGCAACA CGAATTGCTT GGTTTGATTC CAAGCGTCAT CACGTGGTTC 180
AGGCAGCTTT GAAACTGCGT A 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP12 site (N is G or A)
<222>(1)..(201)
<400>12
CCGGCATCGC TACATGAGCA TTAGCCTACA CTCGAGGCGT AGCCAATGCT ATACGCGGCG 60
ATGTCCACCA TACACCGGCA AAAGAAAGCA GCGTTATGGG NCTTTCATGG TGGTGGCCGC 120
ATTCTGGTGG TCTGGAATAG GCCCAATGCT GACTCCAATT GATAACTCTC AACGAGAGTA 180
TTCGATAGTA TTGGTATGAA C 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP13 site (N is T or C)
<222>(1)..(201)
<400>13
GTTTTCAGGG TAATCAGAAT GAATTTGGAA GAACAACTCT CAATTTGAAG TTTGAAATTT 60
GAAAGGTTTG ACCAAGATTT TGATTTGTTA GCATATGATT NTGTATCGGA ATTTTTATGA 120
TTTGGTTAGC CCCGTTAGGA TATTTGGGAC TTATGAGCGT GATCGGAATG CATTTTAGAA 180
GTCCGTGGAA GGTTTAGGCT T 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP14 site (N is T or A)
<222>(1)..(201)
<400>14
ATCAAGAAAA CATTTTGACA TTTCTTTTTG ACACTGTTTC TTGGATCGGA GAACATATTT 60
TTCTTCTGCA ACAAATAGAA GACTTTATTC TCTCATATTT NTATTCAAGT AATAGGGAGA 120
AACATAGTCT GTGAGGAATT ATGTATCAAA AAAGGGTATT CCTTTTGAGT GTGTAATAGC 180
CTCAATGAAT TTGTGATGCT T 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP15 site (N is A or G)
<222>(1)..(201)
<400>15
TCAACCTCTT ATGACCAAGA AACCGTAAGG CCTTATATTC ATCAAATTAA GGATGTCGAC 60
CAGCAGCTTT GCCACAGAGA AGAGCATTGG TAGGTATGTC NTATTCGTTT GCTAAACTGG 120
GCGTTGGATT ATAGACCCTT AGGTAAACTT GCTGTCCAAG ATATTGCCTA TCCCACATTG 180
AAGACCTCAA ATTCCACATA C 201
<211>201
<212>DNA
<213> sequence
<221> Gene sequence containing FP16 site (N is G or A)
<222>(1)..(201)
<400>16
CGTATGTTTA ATCGAGTGAG TGATTCGAAC TCGAAGTAGT GTAGCCCGTA GGTGTAATGA 60
TCGAGTGAGT GTTAGCTCGA ACTCAAAATA AAAGTAGCCC NTAGGCTTAA TGATCGAGTC 120
ATTTTTGAGA TATAAACCAA TATGGAAAGG TTGTACCTTA GCAATAGTAC CGTTTTAGAT 180
GTGATACATT CCAACTGCTT G 201
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP01-F
<222>(1)..(30)
<400>17
ACGTTGGATG GAAGGTCGTT CACGAATCGG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP01-R
<222>(1)..(30)
<400>18
ACGTTGGATG GTCCCTAGTA TTCGAGGTTG 30
<211>23
<212>DNA
<213> Artificial sequence
<221> primer FP01-E
<222>(1)..(23)
<400>19
AGTTCACGAA TCGGTATTAA AAA 23
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP02-F
<222>(1)..(30)
<400>20
ACGTTGGATG AGATTTGGGA TGAGGAGGTG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP02-R
<222>(1)..(30)
<400>21
ACGTTGGATG TTGAAATCCC ACCGCTACTG 30
<211>16
<212>DNA
<213> Artificial sequence
<221> primer FP02-E
<222>(1)..(16)
<400>22
AGCACTACCA CCAACC 16
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP03-F
<222>(1)..(30)
<400>23
ACGTTGGATG ATTGAGGGTT ACCGTCAGAG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP03-R
<222>(1)..(30)
<400>24
ACGTTGGATG CTAAACTCCC CAGAATAACG 30
<211>16
<212>DNA
<213> Artificial sequence
<221> primer FP03-E
<222>(1)..(16)
<400>25
CAGATGCAGA GGGACA 16
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP04-F
<222>(1)..(30)
<400>26
ACGTTGGATG CCAAAACTAC CAGATCCACG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP04-R
<222>(1)..(30)
<400>27
ACGTTGGATG TTGCTAGGGA CATAGAGTTG 30
<211>15
<212>DNA
<213> Artificial sequence
<221> primer FP04-E
<222>(1)..(15)
<400>28
CTCTCCACTC GCTCC 15
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP05-F
<222>(1)..(30)
<400>29
ACGTTGGATG ACCCACTCAA GCAGAAGAAC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP05-R
<222>(1)..(30)
<400>30
ACGTTGGATG TGGATGTGTT CTTCCTCCTC 30
<211>21
<212>DNA
<213> Artificial sequence
<221> primer FP05-E
<222>(1)..(21)
<400>31
GGATTCTTGT TGTTGCATGT T 21
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP06-F
<222>(1)..(30)
<400>32
ACGTTGGATG CAACTTGCCA GTAGGCATAG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP06-R
<222>(1)..(30)
<400>33
ACGTTGGATG ATGCATACAC CACATACCTC 30
<211>19
<212>DNA
<213> Artificial sequence
<221> primer FP06-E
<222>(1)..(19)
<400>34
ACCACATACC TCATATTGA 19
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP07-F
<222>(1)..(30)
<400>35
ACGTTGGATG GTTTGCTTGA CTTGAAGCCC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP07-R
<222>(1)..(30)
<400>36
ACGTTGGATG TGCTGAACTA ATGGGAAGTG 30
<211>24
<212>DNA
<213> Artificial sequence
<221> primer FP07-E
<222>(1)..(24)
<400>37
TGAAGAACTT AATTCTCCCA TTAT 24
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP08-F
<222>(1)..(30)
<400>38
ACGTTGGATG GGTCATGACA TGCACTTTGG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP08-R
<222>(1)..(30)
<400>39
ACGTTGGATG ACCTCAGGCA TAAAAGGGTG 30
<211>17
<212>DNA
<213> Artificial sequence
<221> primer FP08-E
<222>(1)..(17)
<400>40
AAGGGTGAAG AGTGAAA 17
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP09-F
<222>(1)..(30)
<400>41
ACGTTGGATG TCTCTATATA CTCCCTTCCG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP09-R
<222>(1)..(30)
<400>42
ACGTTGGATG CACGAGTTTA CCGTCATCAC 30
<211>22
<212>DNA
<213> Artificial sequence
<221> primer FP09-E
<222>(1)..(22)
<400>43
ATCACATATA TGCTCTTTGC CA 22
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP10-F
<222>(1)..(30)
<400>44
ACGTTGGATG TTCCCATGGG ATTGAATCAG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP10-R
<222>(1)..(30)
<400>45
ACGTTGGATG TCAGCAAAAG CGAGTACCAC 30
<211>17
<212>DNA
<213> Artificial sequence
<221> primer FP10-E
<222>(1)..(17)
<400>46
ATACTCATAC GAGGGGA 17
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP11-F
<222>(1)..(30)
<400>47
ACGTTGGATG AGCAGCTGAG GATTGTAACC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP11-R
<222>(1)..(30)
<400>48
ACGTTGGATG TGCACATGTC TGAGGGTTTC 30
<211>24
<212>DNA
<213> Artificial sequence
<221> primer FP11-E
<222>(1)..(24)
<400>49
CCGTTTTCCT AAAGTTACAG CTTT 24
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP12-F
<222>(1)..(30)
<400>50
ACGTTGGATG ACACCGGCAA AAGAAAGCAG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP12-R
<222>(1)..(30)
<400>51
ACGTTGGATG AGTCAGCATT GGGCCTATTC 30
<211>16
<212>DNA
<213> Artificial sequence
<221> primer FP12-E
<222>(1)..(16)
<400>52
GCCACCACCA TGAAAG 16
<211>29
<212>DNA
<213> Artificial sequence
<221> primer FP13-F
<222>(1)..(29)
<400>53
ACGTTGGATG TTTGAAAGGT TTGACCAAG 29
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP13-R
<222>(1)..(30)
<400>54
ACGTTGGATG AGTCCCAAAT ATCCTAACGG 30
<211>25
<212>DNA
<213> Artificial sequence
<221> primer FP13-E
<222>(1)..(25)
<400>55
GATTTTGATT TGTTAGCATA TGATT 25
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP14-F
<222>(1)..(30)
<400>56
ACGTTGGATG TGTTTCTTGG ATCGGAGAAC 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP14-R
<222>(1)..(30)
<400>57
ACGTTGGATG TCACAGACTA TGTTTCTCCC 30
<211>26
<212>DNA
<213> Artificial sequence
<221> primer FP14-E
<222>(1)..(26)
<400>58
ATAGAAGACT TTATTCTCTC ATATTT 26
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP15-F
<222>(1)..(30)
<400>59
ACGTTGGATG AGCTTTGCCA CAGAGAAGAG 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP15-R
<222>(1)..(30)
<400>60
ACGTTGGATG GTCTATAATC CAACGCCCAG 30
<211>20
<212>DNA
<213> Artificial sequence
<221> primer FP15-E
<222>(1)..(20)
<400>61
CCCAGTTTAG CAAACGAATA 20
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP16-F
<222>(1)..(30)
<400>62
ACGTTGGATG GAGTGTTAGC TCGAACTCAA 30
<211>30
<212>DNA
<213> Artificial sequence
<221> primer FP16-R
<222>(1)..(30)
<400>63
ACGTTGGATG TGCTAAGGTA CAACCTTTCC 30
<211>22
<212>DNA
<213> Artificial sequence
<221> primer FP16-E
<222>(1)..(22)
<400>64
CATGACTCGA TCATTAAGCC TA 22
Claims (9)
1. A primer combination for identifying flue-cured tobacco longjiang 911, which is characterized in that: the primer is designed aiming at 16 specific SNP loci flanking sequences of flue-cured tobacco Longjiang 911, gene sequences containing the 16 SNP loci are shown as SEQ ID NO 1-16, and N of the gene sequences SEQ ID NO 1-16 is A, T, T, C, T, A, T, T, A, T, G, A, C, A, G, A in sequence.
2. The primer combination of claim 1, wherein: the primer sequences of the 16 specific SNP sites are shown as SEQ ID NO 17-64.
3. Cured tobacco longjiang 911 identification kit comprising the primer combination according to claim 1 or 2.
4. The primer combination of claim 1 or 2 and the kit of claim 3 are used for identifying the variety of flue-cured Longjiang 911, 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 16 SNP loci in the detected sample are completely consistent with that of the cured tobacco Longjiang 911, judging that the tobacco sample is the cured tobacco Longjiang 911; the genotypes of 16 SNP loci in flue-cured tobacco Longjiang 911 are AA, TT, CC, TT, AA, TT, GG, AA, CC, AA, GG and AA in sequence.
5. The method for identifying cured tobacco Longjiang 911 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 16 SNP sites in a detection sample are completely consistent with that of the cured tobacco Longjiang 911, judging that the tobacco sample is the cured tobacco Longjiang 911; the genotypes of 16 SNP loci in flue-cured tobacco Longjiang 911 are AA, TT, CC, TT, AA, TT, GG, AA, CC, AA, GG 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: 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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| CN201710556620.7A CN107345251B (en) | 2017-07-10 | 2017-07-10 | Primer combination and kit for identifying flue-cured tobacco Longjiang 911, application and identification method |
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