CN107354201A - Primer for identifying flue-cured tobacco cloud and mist 97 combines and kit, application and detection method - Google Patents
Primer for identifying flue-cured tobacco cloud and mist 97 combines and kit, application and detection method Download PDFInfo
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Abstract
The present invention relates to primer combination and kit, application and the detection method for identifying flue-cured tobacco cloud and mist 97, 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 13 for being applied to identification flue-cured tobacco cloud and mist 97 is obtained according to polymorphic SNP site screening between kind, and contrast 13 SNP site flanking sequences that cultivation tobacco reference gene group obtains, SNP site flanking sequence such as SEQ ID NO:Shown in 1~13, based on the primers；Using the primer of design, parting detection is carried out to these sites using Matrix-assisted laser desorption ionization technology.Obtain SNP genotyping results by detecting, identify whether sample is cloud and mist 97, 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 cloud and mist 97, 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 cloud and mist 97, it is Yunnan Province
Using Yun yan85 as female parent, CV87 is that paternal hybrid seed selection forms for tobacco agriculture research institute.On March 10th, 2009 by complete
Tobacco evaluation committee of state authorizes.Plant turriform, averagely pinch plant height 115.3cm, effective 20 or so, leaf, pitch
5.7cm, stem girth 9.7cm；Leaf long oval, leaf color is green, and blade face is slightly wrinkled, and blade tip blunt point, leaf margin is wavy, and master pulse thickness is medium,
Auricle is medium, waist leaf long 73.9cm, wide 30.3cm；Transplanting is put 52 days or so to the flowers are in blossom at center, and growing stage 125 is in left and right.
Cloud and mist 97 is main cultivation flue-cured tobacco cultivars important in China's leaf tobacco production in recent years, and industrial regular supplies important in production of cigarettes
Kind.
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 CN1057234141A 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 it is a kind of be used for identify flue-cured tobacco cloud and mist 97 primer combination, the primer can simply, quickly,
Efficiently identify whether detection sample is flue-cured tobacco cloud and mist 97.
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 cloud and mist 97.
In order to realize the above object the technical solution adopted in the present invention is：
For identifying that the primer of flue-cured tobacco cloud and mist 97 combines, the primer is directed to 13 specificity of flue-cured tobacco cloud and mist 97 respectively
SNP site flanking sequence designs, with specific reference to the corresponding design primer of difference of detection method.This 13 SNP markers are based near
The exclusive specific SNP positions of flue-cured tobacco cloud and mist 97 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~FP13.Gene order comprising this 13 SNP sites such as SEQ ID NO:1~
Shown in 13.
13 specific SNP sites of the flue-cured tobacco cloud and mist 97 of table 1
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 the cultivar identification of flue-cured tobacco cloud and mist 97 and breeding material detect.
The identification kit of flue-cured tobacco cloud and mist 97, 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 the cultivar identification of flue-cured tobacco cloud and mist 97.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 13 SNP sites is complete
It is complete consistent, then judge the tobacco sample for flue-cured tobacco cloud and mist 97.
The genotype of each SNP site in the flue-cured tobacco cloud and mist 97 of table 2
| FP01 | FP02 | FP03 | FP04 | FP05 | FP06 | FP07 | FP08 | FP09 | FP10 | FP11 | FP12 | FP13 |
| TT | AA | AA | AA | AA | AA | TT | AA | AA | TT | TT | CC | 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 cloud and mist 97
The authentication method of flue-cured tobacco cloud and mist 97, 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~FP13 genotype be followed successively by TT, AA, AA, AA, AA, AA, TT,
AA, AA, TT, TT, CC, TT, then judge the tobacco sample for flue-cured tobacco cloud and mist 97.
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 cloud and mist 97 is obtained according to polymorphic SNP site screening between kind
Mark totally 13, 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, the kind Molecular Detection system of reliable flue-cured tobacco cloud and mist 97, provide base for the identification technology system of flue-cured tobacco cloud and mist 97
Plinth 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 cloud and mist 97.Contrast cultivation tobacco refers to base
Because of group 13 SNP site flanking sequences obtained, and based on SNP site flanking sequence design primer, using ground substance assistant laser
Desorption ionization ionization time of flight carries out parting detection to these sites.The SNP genotyping results obtained according to testing result,
If detection sample in SNP marker FP01~FP13 genotype be followed successively by TT, AA, AA, AA, AA, AA, TT, AA, AA, TT, TT,
CC, TT, then judge the tobacco sample for flue-cured tobacco cloud and mist 97.Amount of samples is few during detection, and the cycle is short, and qualification result is accurate, can weigh
Renaturation is good, and 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 the primer of flue-cured tobacco cloud and mist 97 combines, design, bag are marked for the specific SNP site of flue-cured tobacco cloud and mist 97
Include：
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, 97 specific SNP site of flue-cured tobacco cloud and mist is screened.
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 cloud and mist 97.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 cloud and mist 97.
Present invention screening obtains 13 specific SNP markers for being used to identify flue-cured tobacco cloud and mist 97, and 13 SNP markers are
Based on the exclusive specificity of the flue-cured tobacco cloud and mist 97 that China tobacco main breed full-length genome SNP genotyping results screen in recent years
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；Described 13
Individual SNP marker is FP01-FP13, shown in specific site information Summary table 1.
3rd, for identifying the primer Combination Design of flue-cured tobacco cloud and mist 97.
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
The identification kit of flue-cured tobacco cloud and mist 97：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 of flue-cured tobacco cloud and mist 97, 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、(1μ
M) 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 detecting the testing result and cloud of 13 SNP sites in sample
The finger-print result of cigarette 97 is completely the same, then kind to be identified is flue-cured tobacco cloud and mist 97.
The MassARRAY molecular weight based on Matrix-assisted laser desorption ionization technology that the present invention uses
13 SNP sites that array Platform can be directed to design 13 weight PCR reactions and genotype detection, and experimental design is flexible, genotyping result
Accuracy is high.13 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 | |
| Sample 1 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 2 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 3 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 4 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 5 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 6 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 7 | TT | AA | AA | AA | AA | AA | TT | AA | AA | TT | TT | CC | TT |
| Sample 8 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 9 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 10 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 11 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 12 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 13 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 14 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 15 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 16 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 17 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 18 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 19 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 20 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 21 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 22 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 23 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
| Sample 24 | GG | GG | CC | GG | GG | GG | CC | GG | CC | CC | CC | TT | CC |
As shown in Table 4, there was only the SNP site testing result of sample 7 and the finger-print result of cloud and mist 97 in 24 parts of samples
It is completely the same, illustrate that the detection method has specificity to flue-cured tobacco cloud and mist 97.
<110>Zhengzhou Tobacco Research Institute of CNTC
<120>Primer for identifying flue-cured tobacco cloud and mist 97 combines and kit, application and detection method
<160> 52
<170> SIPOSequenceListing 1.0
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP01
<400> 1
agcctaccgc atttcctgtt agcacattca ttcatccttg tgtcatatct acatactgat 60
gcatattaag gtgtgaatag gatattaccc agccatttta ngcaactttc cacattgtca 120
tttactcgtt acttgtaccc cctactagat gttataacct agtcaatatg cttactgaaa 180
gatataccat tttttagagg a 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP02
<400> 2
tgaggtcccg ggggcctcgg ggtgatttcg gagagttgtc ggaagaattg gaagtggttt 60
gaagcagctg aagctgctga tctctatcat aaccacacct ntggttgggt ctcgtagctg 120
tggagccgca gaagcagcgg agtcatcaca gaagcggaaa ggggagatgc cagcagggac 180
cgcagaagcg ggagcatacc a 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP03
<400> 3
actcaaagtt tttactattc ttgttttcct tcaaaaaatc cctctgcttc acaatctccg 60
catccaagtt ccttgagcaa atcatgtatc ttcttctaat ntttcttccc ttgttctcca 120
atggctaaaa catccaaaat cgtgccgcaa aaagaaactg cgtcttcgtc aaggcctgtc 180
ggcgtcggag ctgcggcaga a 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP04
<400> 4
ttgccttaat tgtagtcatt ttctcagtca tgatgttaca tccgtgtatt atatctctgt 60
ctatgtgcat tctttaatac gtttcccaca tgataatgat ntccttatat gtgtgacagg 120
gttcgagctg agtattgtga tatgtggaca tttgagactt gagctgttga tgattgatct 180
tgggccatag agccgatttg a 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP05
<400> 5
ttgaggtcat cctgggcatg gactggttat ccttatatca cgtcatcctt gattgtcatg 60
ccaagatagt tacgttagaa atgtcagagt tctcaagatt ngagtggaag ggttcctcca 120
ttagtacatc tagtcgggtt atctctttta tgaaggctct acatatagtc gagaagggtt 180
gtttggctta tctagcttat g 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP06
<400> 6
gtagcccggt ccggatttcg gagggtccat tcttctatct ttataacccg accctatctc 60
tataaataga cgtaagggtc catttttaag gggaatatct natagtttta gagagaggaa 120
gtgccctaga gtgagagtag aaatccttaa gctaattgtt catcaaaatc ttgctcaagc 180
cttgaaattc aacaagaaag c 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP07
<400> 7
aaccctcatt ggaagtcgcc tgatcacagc ctcttgtgtt atctttccat cccagctata 60
ttgcatgtca agtgtcttct tcttactttt gtttgaacca nttctcaagt atgtcggtga 120
aagatctcct ttcctagcta cgattgtaaa ggctgcatct agcatttctt cctcgtcccc 180
ttcttcgtag aaaccttctc c 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP08
<400> 8
ttcttattta tggccattag tgtagccttt aatggatatg atatttcttc attaggtgtt 60
tattctttat tattaaactg gcatacgtta atttgaactc ngtttgtatc aaagtacgct 120
ctttactctc tctagaacag cggtaactaa cggctacccc ctagcgagta gccacacgcg 180
cccccactat ggatccacca g 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP09
<400> 9
gaagaaagcc aaccgttgta gtcaatgcgg aaaccggcag gaacaacgcg catggtatgt 60
cttggcatct cgtacccatt tgaaagaaca ttgaaggaag naggagttca ggatcgtttc 120
ttatcatttc ttgccgcttt cactctatga aacatggaga ctatctgtat acggcaaatt 180
cggggccccg atttccccgt c 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP10
<400> 10
gtggatttca gagattaatc atctaatatt gatgtcttaa tggcctcacc atcatcatct 60
tgacaaaact tcacatctag tttcagtttt atcaaagttt ntacggcttg cttctctcag 120
gtacctaaag gatgggacaa actttccatc tctcttatct gcgtggaaac agggaaaaca 180
gtcagcaaat cagggaaagc c 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP11
<400> 11
agcatcaaat aatgccgtac gagacaaacc cggtcgataa aggtcgaatc tttaatcaaa 60
acgcaaagtc aaccagaaag gcaaacccat gaccgcacct nagtacccca caaaacttat 120
aaattccgat aacccattca attatgagtc caaccatact aatttcactc aaatccaact 180
ccgaatcgat gttcaaaact c 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP12
<400> 12
gtgagtccaa ctatgcaaaa atgaaatttg atagaatgtg gttccaaatt tatcataaaa 60
atatacatta aaaggaaagc atttgctgtt ggttaacacg nccgatcagt ttgttgctca 120
gttgcatgaa ctttaaatct ggtgtacaac attcctaata atgagagcgt ttgctaaaga 180
tggttaggta gaatggaagt a 201
<211> 201
<212> DNA
<213>Sequence
<221>Gene order comprising FP13
<400> 13
ctaccttcaa agcaacaaca tgttcattca atccggacaa ctctgcctcc agatcttgga 60
tccgctcatc taatctcact accttgagca ccgacatctc nacttcaata tctcgctcag 120
acctacaaac acggagagca tcatctaggg ctgctgcttc agatatggca gccaccctgc 180
ctttctcggc acgagccaat t 201
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP01-F
<400> 14
acgttggatg gaataggata ttacccagcc 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP01-R
<400> 15
acgttggatg ggttataaca tctagtaggg 30
<211> 21
<212> DNA
<213>Artificial sequence
<221> FP01-E
<400> 16
ggatattacc cagccatttt a 21
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP02-F
<400> 17
acgttggatg tgaagctgct gatctctatc 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP02-R
<400> 18
acgttggatg ttccgcttct gtgatgactc 30
<211> 22
<212> DNA
<213>Artificial sequence
<221> FP02-E
<400> 19
catctctatc ataaccacac ct 22
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP03-F
<400> 20
acgttggatg aatctccgca tccaagttcc 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP03-R
<400> 21
acgttggatg ttgcggcacg attttggatg 30
<211> 18
<212> DNA
<213>Artificial sequence
<221> FP03-E
<400> 22
ggagaacaag ggaagaaa 18
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP04-F
<400> 23
acgttggatg atctctgtct atgtgcattc 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP04-R
<400> 24
acgttggatg acaatactca gctcgaaccc 30
<211> 22
<212> DNA
<213>Artificial sequence
<221> FP04-E
<400> 25
gggtttccca catgataatg at 22
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP05-F
<400> 26
acgttggatg gtcatgccaa gatagttacg 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP05-R
<400> 27
acgttggatg cccgactaga tgtactaatg 30
<211> 15
<212> DNA
<213>Artificial sequence
<221> FP05-E
<400> 28
ggaacccttc cactc 15
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP06-F
<400> 29
acgttggatg ttataacccg accctatctc 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP06-R
<400> 30
acgttggatg tctagggcac ttcctctctc 30
<211> 21
<212> DNA
<213>Artificial sequence
<221> FP06-E
<400> 31
acttcctctc tctaaaacta t 21
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP07-F
<400> 32
acgttggatg tcccagctat attgcatgtc 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP07-R
<400> 33
acgttggatg ggaaaggaga tctttcaccg 30
<211> 18
<212> DNA
<213>Artificial sequence
<221> FP07-E
<400> 34
caccgacata cttgagaa 18
<211> 29
<212> DNA
<213>Artificial sequence
<221> FP08-F
<400> 35
acgttggatg attattaaac tggcatacg 29
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP08-R
<400> 36
acgttggatg gttaccgctg ttctagagag 30
<211> 19
<212> DNA
<213>Artificial sequence
<221> FP08-E
<400> 37
agcgtacttt gatacaaac 19
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP09-F
<400> 38
acgttggatg tggcatctcg tacccatttg 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP09-R
<400> 39
acgttggatg catagagtga aagcggcaag 30
<211> 17
<212> DNA
<213>Artificial sequence
<221> FP09-E
<400> 40
aacgatcctg aactcct 17
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP10-F
<400> 41
acgttggatg gcctcaccat catcatcttg 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP10-R
<400> 42
acgttggatg ccatccttta ggtacctgag 30
<211> 17
<212> DNA
<213>Artificial sequence
<221> FP10-E
<400> 43
gagagaagca agccgta 17
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP11-F
<400> 44
acgttggatg agaaaggcaa acccatgacc 30
<211> 31
<212> DNA
<213>Artificial sequence
<221> FP11-R
<400> 45
acgttggatg gttggactca taattgaatg g 31
<211> 20
<212> DNA
<213>Artificial sequence
<221> FP11-E
<400> 46
ggggacccat gaccgcacct 20
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP12-F
<400> 47
acgttggatg aggaaagcat ttgctgttgg 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP12-R
<400> 48
acgttggatg agttcatgca actgagcaac 30
<211> 16
<212> DNA
<213>Artificial sequence
<221> FP12-E
<400> 49
gctgttggtt aacacg 16
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP13-F
<400> 50
acgttggatg ggatccgctc atctaatctc 30
<211> 30
<212> DNA
<213>Artificial sequence
<221> FP13-R
<400> 51
acgttggatg tctccgtgtt tgtaggtctg 30
<211> 18
<212> DNA
<213>Artificial sequence
<221> FP13-E
<400> 52
tgagcgagat attgaagt 18
Claims (10)
1. the primer combination for identifying flue-cured tobacco cloud and mist 97, it is characterised in that：The primer is directed to 13 spies of flue-cured tobacco cloud and mist 97
Different in nature SNP site flanking sequence design, such as SEQ ID NO of the locus gene sequence comprising 13 SNP:Shown in 1~13.
2. primer combination according to claim 1, it is characterised in that：The primer sequence of 13 specific SNP sites
Such as SEQ ID NO:Shown in 14~52.
3. include the identification kit of flue-cured tobacco cloud and mist 97 of primer as claimed in claim 1 or 2 combination.
4. kit is in terms of the cultivar identification of flue-cured tobacco cloud and mist 97 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 cloud and mist 97 of 13 SNP sites it is completely the same, judge that the tobacco sample is
Flue-cured tobacco cloud and mist 97；In flue-cured tobacco cloud and mist 97 13 SNP site FP01~FP13 genotype successively TT, AA, AA, AA, AA, AA, TT,
AA、AA、TT、TT、CC、TT。
6. using the method for primer as claimed in claim 2 combination identification flue-cured tobacco cloud and mist 97, 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~FP13 genotype be followed successively by TT, AA, AA, AA, AA, AA, TT, AA, AA,
TT, TT, CC, TT, then judge the tobacco sample for flue-cured tobacco cloud and mist 97.
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.
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| Application Number | Priority Date | Filing Date | Title |
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|---|---|---|---|
| CN201710556639.1A CN107354201B (en) | 2017-07-10 | 2017-07-10 | Primer combination and kit for identifying flue-cured tobacco yunyan 97, application and detection method |
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| CN111411167A (en) * | 2020-04-27 | 2020-07-14 | 国家烟草质量监督检验中心 | DNA fingerprint atlas database of tobacco variety and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111411167A (en) * | 2020-04-27 | 2020-07-14 | 国家烟草质量监督检验中心 | DNA fingerprint atlas database of tobacco variety and application thereof |
| CN112410452A (en) * | 2020-04-27 | 2021-02-26 | 国家烟草质量监督检验中心 | DNA fingerprint of Yunyan 87 and application thereof |
| CN112481403A (en) * | 2020-04-27 | 2021-03-12 | 国家烟草质量监督检验中心 | DNA fingerprint of Zhongyan 103 and application thereof |
| CN112501334A (en) * | 2020-04-27 | 2021-03-16 | 国家烟草质量监督检验中心 | DNA fingerprint spectrum of Ningxiang sun-cured tobacco and application thereof |
| CN111411167B (en) * | 2020-04-27 | 2022-10-11 | 国家烟草质量监督检验中心 | DNA fingerprint spectrum library of tobacco variety and application thereof |
| CN112501334B (en) * | 2020-04-27 | 2022-10-11 | 国家烟草质量监督检验中心 | DNA fingerprint spectrum of Ningxiang sun-cured tobacco and application thereof |
| CN112410452B (en) * | 2020-04-27 | 2022-10-14 | 国家烟草质量监督检验中心 | DNA fingerprint of Yunyan 87 and application thereof |
| CN112481403B (en) * | 2020-04-27 | 2022-10-14 | 国家烟草质量监督检验中心 | DNA fingerprint of Zhongyan 103 and application thereof |
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| Publication number | Publication date |
|---|---|
| CN107354201B (en) | 2019-12-27 |
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