CN107354203A - Primer for identifying flue-cured tobacco Bi Na 1 combines and kit, application and detection method - Google Patents
Primer for identifying flue-cured tobacco Bi Na 1 combines and kit, application and detection method Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C12Q2600/156—Polymorphic or mutational markers
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The present invention relates to primer combination and kit, application and the detection method for identifying flue-cured tobacco Bi Na 1, belong to tobacco bred identification technology field.The present invention carries out full-length genome SNP partings using tobacco 420K high density SNP chip to China tobacco main breed in recent years, a set of specific SNP marker totally 15 for being applied to identification flue-cured tobacco Bi Na 1 is obtained according to polymorphic SNP site screening between kind, and contrast 15 SNP site flanking sequences that cultivation tobacco reference gene group obtains, its sequence such as SEQ ID NO:Shown in 1~15, based on the primers；Using the primer of design, parting detection is carried out to these sites using Matrix-assisted laser desorption ionization technology.SNP genotyping results are obtained according to testing result, identify whether sample is Bi Na 1, detection method of the invention when detecting required detection sample size it is few, detection flux is high, and qualification result is accurate.
Description
Technical field
The present invention relates to primer combination and kit, application and the detection method for identifying flue-cured tobacco Bi Na 1, belong to cigarette
Grass product kind identification technology field.
Background technology
Tobacco is a kind of important industrial crops, is the primary raw material of production of cigarettes.Flue-cured tobacco cultivars Bi Na 1 is Guizhou Province
Bijie City company of tobacco company uses the systematic breeding method flue-cured tobacco that seed selection forms from flue-cured tobacco cultivars Yun yan85 natural mutant
New varieties, authorized in October, 2012 by national Guizhou Province of tobacco evaluation committee tobacco bred authorization group.Plant type is in
Tower, it is in cartridge type, plant height 210cm or so after topping, topping plant height 150cm or so, the natural number of sheets 32 or so can picking leaves number
24~26,9~10cm of stem girth, pitch 4.5cm, waist leaf long 78.2cm, wide 29.4cm, the long 61.5cm of top, wide 21.7cm；Leaf
Shape oblong, leaf color green, auricle is big, and blade tip is tapering, corolla pink；Field growing is neat, and growth potential is stronger, tobacco leaf point
It is yellow good that layer falls, easily baking；Transplanting is put 72 days or so to the flowers are in blossom at center, growing stage 135 days or so.Bi Na 1 is in recent years
Important main cultivation flue-cured tobacco cultivars in China's leaf tobacco production, and industrial general types important in production of cigarettes.
SNP (single nucleotide polymorphism, SNP) is primarily referred to as on genome
By the DNA sequence polymorphism that single nucleotide acid variation is caused.Compared with traditional molecular labeling, SNP marker have density it is high,
Have a very wide distribution, the advantages that parting is simple.With whole genome sequence identification technology and the SNP chip typing method of automation
Development, carry out genetic diversity Journal of Sex Research using extensive, high flux SNP chip and become increasingly prevalent.Countries tobacco
Cara gene development and design is informed against a tobacco high density SNP chip (420K Tobacco SNP array), is covered big absolutely
Most Tag SNP sites are simultaneously uniformly distributed whole tobacco gene group, are to carry out heredity to tobacco bred from full-length genome level
Study on Diversity is provided convenience.Genetic diversity using tobacco high density SNP chip labelling technique to tobacco main breed
Analyzed, the specific SNP marker for obtaining specific main breed can be screened.Based on substance assistant laser desorpted ionized
It is anti-that the MassARRAY molecular weight array Platform of ionization time of flight can be directed to the SNP site design weight of highest up to 40 PCR
Should and genotype detection, experimental design is very flexible, and genotyping result accuracy is high.According to using needs, for tens of to hundreds of
When individual SNP site carries out hundreds of to thousands of parts of pattern detections, MassARRAY has high cost performance.Particularly suitable for limited
The SNP site of quantity carries out extensive parting detection.
Kind is the basis of sound tobacco raw material production, is to influence one of most important factor of quality of tobacco.According to《China
People's republic's seed law》With《Law on Monopoly of Tabacoo》, to ensure the stability of leaf tobacco production and sustainable development, it is necessary to from warp
National tobacco evaluation committee examines (recognizing) fixed kind, forbids to plant bad sundry goods kind.In addition, tobacco industry production and product
Exploitation it is also proposed strict requirements to specific tobacco variety.At present, China be faced with tobacco main breed genetic background it is narrow
It is narrow, on morphology similarity degree height be difficult to differentiate between differentiate the problem of.The detection method of tobacco bred is mainly field trapping test
Method.But this method is present that field planting scale is big, discriminating project it is more (diagnostic character be related to plant height, the number of sheets, pitch, leaf length,
Leaf width, cauline leaf angle etc.), the cycle length (cross over tobacco different growing), differentiate difficulty greatly (characteristic index difference is small), homogeneity
Shape has differences the deficiencies of (affected by environment) between year.Tobacco bred is protected, the monitoring of seed purity, true and false detection of tobacco leaf etc.
Aspect, all there is an urgent need to tobacco DNA identity authentications are carried out from molecular level.
Patent CN105734141A discloses a kind of molecular biology method for identifying tobacco bred purity, including：Respectively
Extraction control and the genome DNA of tobacco bred to be measured, the full genome of appropriate depth is carried out using newest sequencing technologies to it
Group sequencing, sequence assembly, assembling and full base are carried out to them using bioinformatics means based on tobacco gene group reference sequences
Because a group sequence compares, the two base difference of statistics, purity percentage of the tobacco bred to be measured relative to control tobacco bred is calculated
Than.This method is not by environmental condition and seasonal effect, and accurately and reliably, can make a variation qualification result water from minimum hereditary unit single base
The flat accurate purity for differentiating tobacco bred, for tobacco bred parent purification and authenticity.But this method complex operation,
Testing cost is very high.
The content of the invention
It is an object of the invention to provide a kind of primer combination for being used to identify flue-cured tobacco Bi Na 1, the primer can simply, soon
Speed, efficiently identify whether detection sample is flue-cured tobacco Bi Na 1.
It is a further object to provide the identification kit combined comprising above-mentioned primer.
Present invention also offers the combination of above-mentioned primer and the application of identification kit.
The present invention also provides a kind of method that can simply, fast and efficiently identify flue-cured tobacco Bi Na 1.
In order to realize the above object the technical solution adopted in the present invention is：
For identifying that flue-cured tobacco Bi Na 1 primer combines, the primer is directed to flue-cured tobacco Bi Na 1 15 specificity respectively
SNP site flanking sequence designs, with specific reference to the corresponding design primer of difference of detection method.This 15 SNP markers are based near
Specific SNP positions exclusive flue-cured tobacco Bi Na 1 that Nian Lai China tobacco main breed full-length genome SNP genotyping results screen
Point, its physical location are that the whole genome sequence based on the cultivation big gold dollar of tobacco bred safflower compares what is determined, specific site letter
Cease as shown in table 1 below, be respectively designated as FP01~FP15.Gene order comprising this 15 SNP sites such as SEQ ID NO:1~
Shown in 15.
1 flue-cured tobacco Bi Na of table 1 15 specific SNP sites
The detection method can use SNP classics detection method such as PCR-RFLP, single-strand conformation polymorphism (SSCP), denaturation ladder
Gel electrophoresis (DGGE), allele specific pcr (AS-PCR) method are spent, or uses SNPs high fluxs detection method such as DNA sequencing
Method, biochip technology, denaturing high-performance chromatography (DHPLC), Taqman sonde methods, SNap shot methods, MassARRAY point
Son amount array technique, to realize that flue-cured tobacco Bi Na 1 cultivar identification and breeding material detect.
No. 1 identification kit of flue-cured tobacco Bi Na, in addition to being combined comprising above-mentioned primer, PCR buffer solutions, MgCl can also be included2、
DNTPs, PCR Enzyme, SAP enzymes, SAP buffer solutions, iPLEX buffer solutions, iPLEX termination mix, iPLEX
Enzyme, water etc..
The application of above-mentioned primer combination or kit in terms of No. 1 cultivar identification of flue-cured tobacco Bi Na.Specially：Take tobacco sample
Genomic DNA carries out SNP parting detections, if the result in detection sample shown in the genotype and table 2 below of 15 SNP sites is complete
It is complete consistent, then judge the tobacco sample for flue-cured tobacco Bi Na 1.
The genotype of each SNP site in 2 flue-cured tobacco Bi Na of table 1
| FP01 | FP02 | FP03 | FP04 | FP05 | FP06 | FP07 | FP08 | FP09 | FP10 | FP11 | FP12 | FP13 | FP14 | FP15 |
| GG | AA | TT | GG | TT | CC | TT | AA | TT | TT | CC | TT | CC | AA | TT |
Present invention preferably employs Matrix-assisted laser desorption ionization technology (MassARRAY molecular weight battle arrays
Row platform), and two amplimers and an extension are designed for each site based on Assay Design Suit (Agena)
Primer, primer combination are as shown in table 3 below.
The primer combination of specific SNP site design of the table 3 based on flue-cured tobacco Bi Na 1
The authentication method that flue-cured tobacco Bi Na 1, comprises the following steps：
1) SNP site multiplexed PCR amplification reacts
Using tobacco sample genomic DNA as template, it is anti-that the amplimer in being combined using primer carries out multiplexed PCR amplification
Should, obtain PCR primer；
2) SAP enzyme reactions
Remaining dNTP and primer in PCR primer are removed with SAP enzymes, obtains reaction product；
3) single base extension
Extension primer is added in the reaction product and carries out single base extension, obtains extension products；
4) genotype detection and result judgement
Extension products are pre-processed, SNP is carried out using Matrix-assisted laser desorption ionization technology
Genotype detection, if detection sample in SNP marker FP01~FP15 genotype be followed successively by GG, AA, TT, GG, TT, CC, TT,
AA, TT, TT, CC, TT, CC, AA, TT, then judge the tobacco sample for flue-cured tobacco Bi Na 1.
Multiplexed PCR amplification, which reacts, in step 1) is：Reaction system：The μ L of 10 × PCR buffer solutions 0.5,25mM MgCl2 0.4μ
L, the μ L of 25mM dNTPs 0.1, μ L of 5U/ μ L PCR Enzyme 0.2, the μ L of mixed liquor 1 of 1 μM of amplimer, 10ng/ μ L tobaccos
The μ L of sample genomic dna 1, water complement to 5 μ L；Reaction condition is：95℃2min；95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min, 45
Individual circulation；72℃5min.
SAP enzyme reactions are in step 2)：μ L and 10 × SAP buffer solutions of 1.7U/ μ L SAP 0.3 are added in PCR primer
0.17 μ L, water complement to 7 μ L；Reaction condition is：37 DEG C of 40min, 85 DEG C of 5min.
Single base extension is in step 3)：Add in the reaction product μ L of 10 × iPLEX buffer solutions 0.2,10 ×
The μ L of iPLEX termination mix 0.2, the μ L of 33U/ μ L iPLEX Enzyme 0.041 and 1 μM of extension primer mixed liquor
0.94 μ L, water complement to 9 μ L；Reaction condition is：94℃30s；[94 DEG C of 5s, (52 DEG C of 5s, 80 DEG C of 5s) 5 circulation] 40 are followed
Ring；72℃3min.
Pre-processed in step 4) and be：Water 41 μ L, clean resin 15mg are added in extension products, desalination is carried out after mixing and is gone
The anti-tampering processing of ion, centrifuging and taking supernatant are standby.
SNP genotype detections are in step 4)：Will be upper using MassARRAY Nanodispenser RS1000 point sample instruments
Chip is placed in MassARRAY Typer Workstation MA4 by clear liquid o'clock on 384 point SpectroCHIP chips, is used
MALDI-TOF (substance assistant laser desorpted ionized flight time) mass spectrograph scans chip, obtains SNP genotype call results.
All primers are equivalent in the primer mixed liquor in the present invention.
Beneficial effects of the present invention：
The present invention carries out full-length genome using tobacco 420K high density SNP chip to China tobacco main breed in recent years
SNP partings, a set of specific SNP for being applied to identification flue-cured tobacco Bi Na 1 is obtained according to polymorphic SNP site screening between kind
Mark totally 15, specific site information sees the above table 1, and designs corresponding primer according to site information, and particular sequence is shown in Table 3, and profit is adopted
SNP parting detections are carried out to candidate sample with Matrix-assisted laser desorption ionization technology, establish it is a set of it is easy,
Fast, efficiently, No. a kind Molecular Detection system of reliable flue-cured tobacco Bi Na, provided for the flue-cured tobacco Bi Na identification technology systems of No. 1
Basis and foundation.
The present invention uses the MassARRAY molecular weight battle arrays based on Matrix-assisted laser desorption ionization technology
Row platform can be directed to SNP site design highest up to 40 weight PCR reactions and genotype detection, and experimental design is very flexible, point
Type result accuracy is high.According to using needs, hundreds of to thousands of parts of pattern detections are carried out to hundreds of SNP sites for tens of
When, MassARRAY has high cost performance.Particularly suitable for carrying out extensive parting detection to the SNP site of limited quantity.
The present invention carries out full-length genome SNP using tobacco 420K high density SNP chip to China's difference flue-cured tobacco main breed
Parting, screening obtain a set of specific SNP marker for being applied to identification flue-cured tobacco Bi Na 1.Contrast cultivation tobacco reference
15 SNP site flanking sequences that genome obtains, and based on SNP site flanking sequence design primer, swashed using Matrix-assisted
Photodesorption ionization time of flight mass spectrometry technology carries out parting detection to these sites.The SNP parting knots obtained according to testing result
Fruit, if detection sample in SNP marker FP01~FP15 genotype be followed successively by GG, AA, TT, GG, TT, CC, TT, AA, TT, TT,
CC, TT, CC, AA, TT, then judge the tobacco sample for flue-cured tobacco Bi Na 1.Amount of samples is few during detection, and the cycle is short, qualification result
Accurately, favorable repeatability, detection flux is high, there is good application prospect.
Embodiment
With reference to specific embodiment, the present invention is described in further detail.All primers are by China in the present embodiment
Big gene chemical synthesis.
Embodiment 1
For identifying that flue-cured tobacco Bi Na 1 primer combines, design is marked for the flue-cured tobacco Bi Na specific SNP sites of No. 1,
Including：
1st, tobacco main breed full-length genome SNP partings detect.
China tobacco main breed in recent years is carried out using 420K Tobacco SNP array (Affymetrix) complete
Genome scanning, countries tobacco cara gene Gene Titan chip platforms (Affymetrix) are relied on to carry out sample DNA
SNP partings.Product random fragment is turned to 25 to the fragment between 125bp by tobacco sample DNA after whole genome amplification.Piece
Section carries out resuspension after purification, is hybridized with 420K Tobacco SNP array.Each SNP is sent out by chip surface
Raw double-colored coupled reaction carries out discriminating connection.Stringency washes are carried out after the completion of crossover process to remove non-specific binding.
After the completion of coupled reaction, chip completes the steps such as dyeing washing on Gene Titan multichannels automation chip operation station, most
After be scanned and output result.The data that chip analysis obtains are handled, obtain different cultivars SNP genotyping results.
2nd, the specific SNP site screenings of flue-cured tobacco Bi Na 1.
According to chip system data staging and type of recommendation, Poly high resolution and Mono high are only chosen
Resolution two types site data, obtain high quality SNP genotyping results after filtering, be retained in all detection kinds
Call rate are 100% site, and further screening removes the site for occurring heterozygosis parting in any kind, finally obtains
Obtain homozygous SNP site in all detection kinds.Screening obtains the specific SNP site in flue-cured tobacco Bi Na 1.Due to cigarette
A large amount of repetitive sequences in grass be present, it is each to SNP site flank to avoid detection primer from non-specific amplification occur in designing
200bp sequences carry out blast with reference gene group and compared, the site without height similar sequences in screening-gene group.With reference to SNP positions
Distribution situation on chromosome is put, a site is selected on the chromosome for exist pleomorphism site, chromosomal polymorphism is preferable
Select specific SNP marker of two sites as flue-cured tobacco Bi Na 1.
Present invention screening obtains 15 specific SNP markers for being used to identify flue-cured tobacco Bi Na 1,15 SNP markers
It is No. 1 exclusive spy of flue-cured tobacco Bi Na screened based on China tobacco main breed full-length genome SNP genotyping results in recent years
Different in nature SNP site, its physical location are that the whole genome sequence based on the cultivation big gold dollar of tobacco bred safflower compares what is determined；Institute
It is FP01-FP15 to state 15 SNP markers, shown in specific site information Summary table 1.
3rd, for identifying flue-cured tobacco Bi Na 1 primer Combination Design.
The specific SNP site obtained to screening carries out chromosome mapping in reference gene group, and acquisition includes these SNP
The upstream and downstream sequence in site.Based on Assay Design Suit (Agena) for each site design two amplimers and one
Bar extension primer, primer sequence is as shown in Summary table 3.
Embodiment 2
No. 1 identification kit of flue-cured tobacco Bi Na：Buffered comprising amplimer mixed liquor in embodiment 1 (1 μM) 5mL, 10 × PCR
Liquid 5mL, 25mM MgCl24mL, 25mM dNTPs 2mL, 5U/ μ L PCR Enzyme 2mL, 1.7U/ μ L SAP3mL, 10 ×
SAP buffer solutions 3mL, 10 × iPLEX buffer solution 2mL, 10 × iPLEX termination mix 2mL, 33U/ μ L iPLEX
Enzyme 1mL, extension primer mixed liquor (1 μM) 5mL, water 200mL in embodiment 1.
Embodiment 3
The authentication method that flue-cured tobacco Bi Na 1, comprises the following steps：
1st, testing sample DNA is extracted：Sample fresh leaf tissue is gathered, utilizes Gene Pure Neway Plant
Genomic DNA Kit kits (Gene Answer) extraction sample gene group DNA；Utilize nucleic acid-protein analyzer
NanoDrop ND-2000 (ThermoFisher Scientific) detect DNA concentration, and it is standby that DNA is diluted into 10ng/ μ L.
2nd, MassARRAY is detected：Operating method requires to carry out according to MassARRAY system platforms (Agena), and reaction utilizes
IPLEX Gold Reagent Kit kits (Agena) are carried out, and are specially：
1) SNP site multiplexed PCR amplification reacts：
Using testing sample genomic DNA as template, multiplexed PCR amplification reaction is carried out using amplimer in embodiment 1, is obtained
PCR primer；
Reaction system is：μ L of PCR buffer solutions (10 ×) 0.5, MgCl2(25mM)0.4μL、dNTPs(25mM)0.1μL、PCR
Enzyme (5U/ μ L) 0.2 μ L, mixed liquor (1 μM) 1 μ L of amplimer, genomic DNA (10ng/ μ L) 1 μ L, water complement to 5 μ
L.Reaction condition is：95℃2min；95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min, 45 circulations；72℃5min.
2) SAP enzyme reactions：
Shrimp alkaline phosphotase (shrimp alka-line phosphatase, SAP) removes dNTP in PCR primer；In step
SAP (1.7U/ μ L) 0.3 μ L, μ L of SAP buffer solutions (10 ×) 0.17, water are added in rapid 1) PCR primer and complements to 7 μ L；Reaction condition
For：37 DEG C of 40min, 85 DEG C of 5min.
3) single base extension：
Extension is carried out using iPLEX Reagent Kit：IPLEX buffer solutions are added in the product that step 2) obtains
(10×)0.2μL、iPLEX termination mix(10×)0.2μL、iPLEX Enzyme(33U/μL)0.041μL、1μM
μ L of mixed liquor 0.94, the water of extension primer complement to 9 μ L；Reaction condition is：94℃30s；[94℃5s、(52℃5s、80℃5s)
5 circulations] 40 circulations；72℃3min；
4) genotype detection：
The μ L of water 41 are added in step 3) product, clean resin 15mg (96 orifice plate) is reverse to shake up 15min progress desalinations
The anti-tampering processing of ion；3200g centrifuges 5min；Take supernatant standby.Utilize MassARRAY Nanodispenser RS1000 points
Sample instrument is to the supernatant point sample instrument of step 4) extension products by sample point to 384 point SpectroCHIP (chip).Chip is put
To in MassARRAY Typer Workstation MA4, using MALDI-TOF (during substance assistant laser desorpted ionized flight
Between) mass spectrograph scanning chip, scanning result is with the software analysis of Typer 4.0 and exports result.
3rd, testing result compares
The SNP marker testing result of acquisition is judged, if the testing result of 15 SNP sites is with finishing in detection sample
No. 1 finger-print result of receiving is completely the same, then kind to be identified is flue-cured tobacco Bi Na 1.
The MassARRAY molecular weight based on Matrix-assisted laser desorption ionization technology that the present invention uses
15 SNP sites that array Platform can be directed to design 15 weight PCR reactions and genotype detection, and experimental design is flexible, genotyping result
Accuracy is high.15 SNP sites can be carried out with the detection of 396 parts of samples, detection flux is high simultaneously.
Test example
By taking 24 parts of different cultivars samples as an example, identified using the method in embodiment 3, verify the special of this method
Property, as a result as shown in table 4.Participating in detection sample 1-24 is respectively：The big gold dollar of flue-cured tobacco K326, safflower, Zhongyan-100, it is dark green No. 1,
Yun yan85, cloud and mist 87, cloud and mist 97, cloud and mist 100, NC95, Longjiang 911, Longjiang 981, Qin's cigarette 96, Bi Na 1, Nan Jiang 3；Spices
Smoke, mists and clouds perfume (or spice) Bath agate 1, Yun Xiang 2, Basma；Burley tobaccos E'yan 1, HubeiT3, VAM, Burley-21；Cigar
Beinhart-1000、Havana-10、Florida-301.Participate in detection sample, the big gold dollar of flue-cured tobacco K326, safflower, middle cigarette
100th, dark green No. 1, Yun yan85, cloud and mist 87, cloud and mist 97, cloud and mist 100, Longjiang 911, Longjiang 981, Qin's cigarette 96, Bi Na 1, Nan Jiang 3
Number；Spices smoke, mists and clouds perfume (or spice) Bath agate 1, Yun Xiang 2；Burley tobaccos E'yan 1, HubeiT3 are in recent years in China's leaf tobacco production
The main breed promoted the use of.
4 24 parts of tobacco sample testing results of table
| FP01 | FP02 | FP03 | FP04 | FP05 | FP06 | FP07 | FP08 | FP09 | FP10 | FP11 | FP12 | FP13 | FP14 | FP15 | |
| Sample 1 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 2 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 3 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 4 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 5 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 6 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 7 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 8 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 9 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 10 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 11 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 12 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 13 | GG | AA | TT | GG | TT | CC | TT | AA | TT | TT | CC | TT | CC | AA | TT |
| Sample 14 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 15 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 16 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 17 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 18 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 19 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 20 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 21 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 22 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 23 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
| Sample 24 | AA | GG | CC | CC | CC | TT | GG | CC | CC | CC | GG | CC | TT | TT | CC |
As shown in Table 4, there was only finger-print knot of the SNP site testing result with Bi Na 1 of sample 13 in 24 parts of samples
Fruit is completely the same, illustrates that the detection method has specificity for No. 1 to flue-cured tobacco Bi Na.
<110>Zhengzhou Tobacco Research Institute of CNTC
<120>Primer for identifying flue-cured tobacco Bi Na 1 combines and kit, application and detection method
<160> 60
<170> SIPOSequenceListing 1.0
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP01
<400> 1
gcttatagat atggtataga gtgactaagg tacatgtaaa ggtggcatct ttttataagt 60
atattagtgc ttctataaat tctttcatct cccacatgtt nctttgtgac ttacctactc 120
aatacattct tccatactga cgtccccact gggaatgcta catttcatgt tgcaggcaca 180
gatactcaag ctagtagacc t 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP02
<400> 2
gacatccgcc catgtcatta tttggaaatc gagcaggctt tctttcaact ttcgggaggc 60
gtcgaagctc ctcggattca aaccctctgt gaatacctcg nctgcccatt gataagtgag 120
gattttgact gcttattagc accttttagc ttttatttta gtccaaaatc attgaattgt 180
gttcccgaaa ctaatgaaat t 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP03
<400> 3
agctcgggct cgacccaaac attgaccccg aagtccctca tcgaccagtc ttacacccgg 60
gaagtagggt ttctcggttt atttaactcc ctgattgagc ntgtatcctc tcgtaaaacg 120
cgcccgaaca gtaagccttt aggtttggtt atctccccat tcttagctcg catttcatca 180
ttaaacttca tattcttagc a 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP04
<400> 4
ttattaattt cacattctcg tattacctta gctatgatga ctctccctta tagttaaatg 60
atatttttgg catgttatac acttctataa tttaagaccg naagatccaa attaaaagta 120
gtgtttatat tcttacatgg tgtccagtca aactaccatg cctaaaatgt agtggagtga 180
gcaagatttc ataagaataa c 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP05
<400> 5
aataaagcac atcaaaaata gaacatatat caaaataagg catcatatgc cacaacttgc 60
caaaattaac aatactgaag ttatcaaaac atatatccca ngactcaacc acaatacgaa 120
caagaatctc aataataaca aaatgaacgt gaattactaa acaaggaaag atatctcggt 180
aatcaataac ttcaaataag a 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP06
<400> 6
gaaaatgtat atatactttt gtggtatgat atcactggga tgttggggtt tgcatttaaa 60
gtcattcatt ggcacttaga cctgtaggga agattaagcg ngagtggtta ggtgtatttg 120
ggagtagatt ttagttcctg gttgggttat tctccctgac acaagcactg ccctgggccc 180
tgagaccctt gggaactttg a 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP07
<400> 7
aggtggttat cagaactgta tctatgcttt agcggatcga caggactagg agtcttcacc 60
agacgttgtg ataggtatat tgacaatttg ctcttatgat ncttatacct tgatggatcc 120
aggatctaat ttatcgtata ttaccccatt tgtcgtgggg aagtttggta tagtgcgtgg 180
aattttatgt tatccctttg t 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP08
<400> 8
atctcttcga gggtctttct tcgagatttg cagcgagcat attcttcaac cctccctttg 60
cggtcacgga tccttctcag ctcagcttga gcatcagcag natcctttaa atagattgac 120
atctcccggt gtgccttagt ccgacttatc gctgcttttg cacgagcatc aacgatctca 180
accctgattt ttgaaagctc g 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP09
<400> 9
ggaccgcggt agcttgaatt ccaaaaatga caactctctg aatccttacc accaaagacc 60
gcgataaaag gattgtggtt gtggtgggtc ttctacgtcc ntggtggact ttccgttgta 120
gcgctgcttt gacttagttt tgaatttctg caggtttcac taattttttg agctggttat 180
gacatccttg tccctttttg c 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP10
<400> 10
atgtcattca ttttcattac caaaataaag acaactcctc aattctgaat aacaattttt 60
tctcttgtca agaaatcttg taatatctct acacttaaaa ntagaatgac aggtatctta 120
agcatttgga ttagcggaag taccgtaatt cccattttag gcagtatcgt ctcaataaaa 180
atacgggtct tcctgtaaca a 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP11
<400> 11
gtcatggtct taatttgttg ctaggttcaa aaggctctat acatgcactt aaacttcaat 60
agttagcctt tcctgttata gcttttgtga catccctaga nccattgcct tattcttgct 120
tagttcaaaa ggctcgataa tttcttttag acaggctcat gcattaaaag tccataagct 180
gttggattaa ccacaaattg a 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP12
<400> 12
tattatgcag ttgatattgt gctaagtatt gttctatcac agatggtgag aacgcatgcg 60
gcagatgtac caggcgatag atgagattct cccttcattg ntagaggacg aggtagaggc 120
cgggggaggg ctccagctcc tattagagga cgaaggcgtc ctagagttgc tcccgttgta 180
ccactagtgg atccaatgca g 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP13
<400> 13
gatcacaaca catgtacgag taatcaagtt gtctcacaat ccacctagcc taatagagta 60
ttacatggaa tagctgacga caggaataac attcaatgcc ngatgactca tccgtaagca 120
attctatgac gaatgaactc atccgaactg acgtagagca cacattcaca ttagaccaac 180
aaacggacct caaaccgatt c 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP14
<400> 14
ctgcatgtgt tgcttgattt gttgttactc ctaccccctt cttgaatttg agatctgcat 60
agtatccatg taacttgtaa caattgtcgc tagtgcatcc nttgaaatgc agtagtcgcg 120
ttgcaaattt ttcttcttct gcttgtaatt acgggggccg agtcctccta gtgtttcaga 180
agagtgttgt agcttcattc a 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP15
<400> 15
gagaacgaca acctcgaaaa ctatggagaa aatggtgtgg ttgttccagt tgttggcacg 60
ccaccacaga acccctataa cacacctgga ccgattccaa nagacgcgga tttgcaagac 120
gtgcaatagg tcgacgaaac ctcacatacc gacaggagca tacaccatgg cgaccaacag 180
gaagtccaaa gaaccccacc c 201
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP01-F
<400> 16
acgttggatg gtacatgtaa aggtggcatc 30
<211> 29
<212> DNA
<213>Artificial sequence
<221> FP01-R
<400> 17
acgttggatg tgtattgagt aggtaagtc 29
<211> 20
<212> DNA
<213>Artificial sequence
<221> FP01-E
<400> 18
tgagtaggta agtcacaaag 20
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP02-F
<400> 19
acgttggatg gaagctcctc ggattcaaac 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP02-R
<400> 20
acgttggatg aaaggtgcta ataagcagtc 30
<211> 17
<212> DNA
<213>Artificial sequence
<221> FP02-E
<400> 21
cctctgtgaa tacctcg 17
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP03-F
<400> 22
acgttggatg tcttacaccc gggaagtagg 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP03-R
<400> 23
acgttggatg ctaaaggctt actgttcggg 30
<211> 18
<212> DNA
<213>Artificial sequence
<221> FP03-E
<400> 24
ttaactccct gattgagc 18
<211> 29
<212> DNA
<213>Artificial sequence
<221> FP04-F
<400> 25
acgttggatg ttggcatgtt atacacttc 29
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP04-R
<400> 26
acgttggatg tggtagtttg actggacacc 30
<211> 24
<212> DNA
<213>Artificial sequence
<221> FP04-E
<400> 27
ctacacttct ataatttaag accg 24
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP05-F
<400> 28
acgttggatg atcatatgcc acaacttgcc 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP05-R
<400> 29
acgttggatg gattcttgtt cgtattgtgg 30
<211> 23
<212> DNA
<213>Artificial sequence
<221> FP05-E
<400> 30
aagttatcaa aacatatatc cca 23
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP06-F
<400> 31
acgttggatg cacttagacc tgtagggaag 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP06-R
<400> 32
acgttggatg agggagaata acccaaccag 30
<211> 18
<212> DNA
<213>Artificial sequence
<221> FP06-E
<400> 33
tgtagggaag attaagcg 18
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP07-F
<400> 34
acgttggatg tcaccagacg ttgtgatagg 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP07-R
<400> 35
acgttggatg tagatcctgg atccatcaag 30
<211> 21
<212> DNA
<213>Artificial sequence
<221> FP07-E
<400> 36
gtggatccat caaggtataa g 21
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP08-F
<400> 37
acgttggatg atccttctca gctcagcttg 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP08-R
<400> 38
acgttggatg gataagtcgg actaaggcac 30
<211> 23
<212> DNA
<213>Artificial sequence
<221> FP08-E
<400> 39
agatgtcaat ctatttaaag gat 23
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP09-F
<400> 40
acgttggatg ccaaagaccg cgataaaagg 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP09-R
<400> 41
acgttggatg agtcaaagca gcgctacaac 30
<211> 17
<212> DNA
<213>Artificial sequence
<221> FP09-E
<400> 42
ggacggaaag tccacca 17
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP10-F
<400> 43
acgttggatg ctcttgtcaa gaaatcttgt 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP10-R
<400> 44
acgttggatg tacggtactt ccgctaatcc 30
<211> 26
<212> DNA
<213>Artificial sequence
<221> FP10-E
<400> 45
atcttgtaat atctctacac ttaaaa 26
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP11-F
<400> 46
acgttggatg cctgttatag cttttgtgac 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP11-R
<400> 47
acgttggatg tgcatgagcc tgtctaaaag 30
<211> 21
<212> DNA
<213>Artificial sequence
<221> FP11-E
<400> 48
agcttttgtg acatccctag a 21
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP12-F
<400> 49
acgttggatg gcgatagatg agattctccc 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP12-R
<400> 50
acgttggatg gagcaactct aggacgcctt 30
<211> 17
<212> DNA
<213>Artificial sequence
<221> FP12-E
<400> 51
ctctacctcg tcctcta 17
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP13-F
<400> 52
acgttggatg atggaatagc tgacgacagg 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP13-R
<400> 53
acgttggatg acgtcagttc ggatgagttc 30
<211> 25
<212> DNA
<213>Artificial sequence
<221> FP13-E
<400> 54
cacgccagga ataacattca atgcc 25
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP14-F
<400> 55
acgttggatg acccccttct tgaatttgag 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP14-R
<400> 56
acgttggatg aatttgcaac gcgactactg 30
<211> 20
<212> DNA
<213>Artificial sequence
<221> FP14-E
<400> 57
ccgcgactac tgcatttcaa 20
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP15-F
<400> 58
acgttggatg ccacagaacc cctataacac 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP15-R
<400> 59
acgttggatg gaggtttcgt cgacctattg 30
<211> 16
<212> DNA
<213>Artificial sequence
<221> FP15-E
<400> 60
cctggaccga ttccaa 16
Claims (10)
1. the primer combination for identifying flue-cured tobacco Bi Na 1, it is characterised in that：The primer is directed to 15 of flue-cured tobacco Bi Na 1
Specific SNP site flanking sequence design, such as SEQ ID NO of the gene order comprising 15 SNP sites:Shown in 1~15.
2. primer combination according to claim 1, it is characterised in that：The primer sequence of 15 specific SNP sites
Such as SEQ ID NO:Shown in 16~60.
3. include No. 1 identification kit of flue-cured tobacco Bi Na of primer as claimed in claim 1 or 2 combination.
4. kit is in terms of No. 1 cultivar identification of flue-cured tobacco Bi Na described in primer combination as claimed in claim 1 or 2, claim 3
Application.
5. application according to claim 4, it is characterised in that：Including：Tobacco sample genomic DNA is taken to carry out SNP partings
Detection, if in detection sample genotype and the flue-cured tobacco Bi Na No. 1 number of 15 SNP sites it is completely the same, judge the tobacco sample
For flue-cured tobacco Bi Na 1；In flue-cured tobacco Bi Na 1 15 SNP site FP01~FP15 genotype successively GG, AA, TT, GG, TT,
CC、TT、AA、TT、TT、CC、TT、CC、AA、TT。
6. using the primer as claimed in claim 2 combination identification flue-cured tobacco Bi Na methods of No. 1, it is characterised in that：Including following step
Suddenly：
1) SNP site multiplexed PCR amplification reacts
Using tobacco sample genomic DNA as template, the amplimer in being combined using primer carries out multiplexed PCR amplification reaction, obtains
PCR primer；
2) SAP enzyme reactions
Remaining dNTP and primer in PCR primer are removed with SAP enzymes, obtains reaction product；
3) single base extension
Extension primer is added in the reaction product and carries out single base extension, obtains extension products；
4) genotype detection and result judgement
Extension products are pre-processed, SNP genes are carried out using Matrix-assisted laser desorption ionization technology
Type detect, if detection sample in SNP marker FP01~FP15 genotype be followed successively by GG, AA, TT, GG, TT, CC, TT, AA, TT,
TT, CC, TT, CC, AA, TT, then judge the tobacco sample for flue-cured tobacco Bi Na 1.
7. according to the method for claim 6, it is characterised in that：Multiplexed PCR amplification, which reacts, in step 1) is：Reaction system：
The μ L of 10 × PCR buffer solutions 0.5,25mM MgCl2 0.4μL、25mM dNTPs 0.1μL、5U/μL PCR Enzyme 0.2μL、1
The μ L of mixed liquor 1, the μ L of 10ng/ μ L tobacco samples genomic DNA 1 of μM amplimer, water complement to 5 μ L；Reaction condition is：95
℃2min；95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min, 45 circulations；72℃5min.
8. according to the method for claim 7, it is characterised in that：SAP enzyme reactions are in step 2)：Added in PCR primer
The μ L of 0.3 μ L and 10 × SAP buffer solutions of 1.7U/ μ L SAP 0.17, water complement to 7 μ L；Reaction condition is：37 DEG C of 40min, 85 DEG C
5min。
9. according to the method for claim 8, it is characterised in that：Single base extension is in step 3)：In reaction product
μ L of 10 × iPLEX of middle addition buffer solutions 0.2, the μ L of 10 × iPLEX termination mix 0.2,33U/ μ L iPLEX
The μ L of Enzyme 0.041 and the μ L of the mixed liquor of 1 μM of extension primer 0.94, water complement to 9 μ L；Reaction condition is：94℃30s；94℃
5s, 52 DEG C of 5s, 80 DEG C of 5s, 5 circulations, 40 circulations；72℃3min.
10. according to the method for claim 6, it is characterised in that：Pre-processed in step 4) and be：Water is added in extension products
41 μ L, clean resin 15mg, carry out desalination deionization processing after mixing, centrifuging and taking supernatant is standby；Using point sample instrument by supernatant
Liquid point scans chip with MALDI-TOF mass spectrographs, obtains SNP genotype call results on chip.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710557206.8A CN107354203B (en) | 2017-07-10 | 2017-07-10 | Primer combination and kit for identifying cured tobacco Bina No. 1, application and detection method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710557206.8A CN107354203B (en) | 2017-07-10 | 2017-07-10 | Primer combination and kit for identifying cured tobacco Bina No. 1, application and detection method |
Publications (2)
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| CN107354203B CN107354203B (en) | 2019-12-27 |
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| Application Number | Title | Priority Date | Filing Date |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111898807A (en) * | 2020-07-14 | 2020-11-06 | 云南省烟草农业科学研究院 | Tobacco yield prediction method based on whole genome selection and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013080045A3 (en) * | 2011-11-28 | 2013-08-01 | 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 CN201710557206.8A patent/CN107354203B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013080045A3 (en) * | 2011-11-28 | 2013-08-01 | 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 |
Non-Patent Citations (2)
| Title |
|---|
| 肖炳光等: "利用基因组简约法开发烟草SNP 标记及遗传作图", 《作物学报》 * |
| 董园园等: "一种新的基于单碱基延伸的SNP芯片技术", 《遗传》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111898807A (en) * | 2020-07-14 | 2020-11-06 | 云南省烟草农业科学研究院 | Tobacco yield prediction method based on whole genome selection and application |
| CN111898807B (en) * | 2020-07-14 | 2024-02-27 | 云南省烟草农业科学研究院 | Tobacco leaf yield prediction method based on whole genome selection and application |
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| Publication number | Publication date |
|---|---|
| CN107354203B (en) | 2019-12-27 |
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