CN106957918A - Method and the application of cape jasmine kind are identified using EST SSR primers - Google Patents
Method and the application of cape jasmine kind are identified using EST SSR primers Download PDFInfo
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Abstract
The invention provides a kind of method that utilization EST SSR primers identify cape jasmine kind, including following steps：Extract experimental cultivar sample leaf DNA, carry out pcr amplification reaction, polyacrylamide gel electrophoresis and silver staining and EST SSR spectral band analysis using EST SSR primers.By carrying out EST SSR spectral band analysis after the amplification of 14 pairs of EST SSR primer PCRs, can to carrying out cultivar identification for examination cape jasmine kind and known cape jasmine kind, the cape jasmine Main Cultivars EST SSR finger-prints of structure can as cape jasmine cultivar identification control.This identifies the method for cape jasmine kind using EST SSR molecular markers, cape jasmine kind can fast and effectively be identified, and qualification result is difficult affected by environment, the main breed of fructus gardeniae, water cape jasmine and the gardenia different application type of current establishing in large scale can be gone out by leaf sample precise Identification in cape jasmine plantlet stage.
Description
Technical field
It is more particularly to a kind of to utilize EST-SSR primers mirror the present invention relates to molecular biology molecule labelling technique field
Determine method and the application of cape jasmine kind.
Background technology
Cape jasmine (Gardenia jasminoides Ellis) is that Rubiaceae (Rubiaceae) Gardenia Ellis (Gardenia) is normal
Green shrub, with fruit medicine, tool purging intense heat relieving restlessness, reducing fever and causing diuresis, effect of removing pattogenic heat from the blood and toxic material from the body.Cape jasmine is the traditional large medicine of China
Material, existing more than 2000 years applicating history, main product Jiangxi, Hunan, Chongqing, Sichuan, Hubei, Zhejiang, Anhui, Fujian, Guangxi etc.
Provinces and cities' also common cultivation.Area, more than 26667 hectares, is local important ecological warp during Jiangxi Province Yellow Fructus Gardeniae artificial growth peak
One of Ji industry.Due to the difference of growing environment, add long-term artificial cultivation and seed selection, make its habit, flower, the shape of leaf and
Some variations occur for size, form and dimension of fruit etc., occur in that many excellent Farm cultivars and type, can be big
Cause is divided into dye mast, medicinal mast and views and admires mast three major types type.Current people only come to the different product of cape jasmine with mode of appearance
Type makes a distinction, and Fructus Gardeniae is referred to as fructus gardeniae, and flower single-lobe, fruit head is smaller, and fruit unit price is higher；Dye uses cape jasmine
Be referred to as water cape jasmine, flower single-lobe, fruit head is larger, and pigment content is high, it is unbearably medicinal to make based on dyestuff, fruit unit price compared with
It is low；View and admire cape jasmine and be referred to as gardenia, flower polyphyll, Hua great, result, is not used as ornamental plantation.Distinguished with mode of appearance such as flower, fruits
Cape jasmine kind is limited by plant age, is difficult effectively reliably to make a distinction in cape jasmine Seedling Stage, and gardenia only blooms not result,
The water cape jasmine market price less than the fructus gardeniae market price half, if just it is found not to be after the cape jasmine of plantation yields positive results
Desired kind, certainly will cause greatly loss.
With the development of Protocols in Molecular Biology, cape jasmine kind is identified in gene level using DNA molecular marker
Be possibly realized, relative to phenotypic character identification method, DNA molecular marker technology to the identification more accurate and effective of cape jasmine kind, and
The limitation of plant age is breached, the identification for cape jasmine kind Seedling Stage provides accurately and reliably method.It is not yet relevant at present
It is used for the method and application report that identification is made a distinction to cape jasmine main breed in EST-SSR molecular labelings.
The content of the invention
It is an object of the invention to overcome the shortcoming of above-mentioned prior art, an aspect of of the present present invention provides one kind and utilizes EST-
The method that SSR primers identify cape jasmine kind, can fast and effectively be identified cape jasmine kind.This method uses general
Logical primer is expanded and detected with denaturing polyacrylamide gel electrophoresis, is examined with the Capillary Electrophoresis of fluorescent dye primer
Survey method is easily operated compared to more economical material benefit.
A kind of method that utilization EST-SSR primers identify cape jasmine kind, including：
Step one, the DNA of experimental cultivar sample is extracted；
Step 2, pcr amplification reaction is carried out using EST-SSR primers, and the EST-SSR primers are 14 pairs, and its sequence is such as
Under：
Step 3, Polyacrylamide Gel Electrophoresis；
Step 4, EST-SSR spectral band analysis build molecular marking fingerprint.
Beneficial effects of the present invention are：This identifies the method for cape jasmine kind using EST-SSR primers, can be to cape jasmine kind
Fast and effectively identified, and qualification result is difficult to be influenceed by plant age, can accurately distinguish Seedling Stage cape jasmine kind,
Pigment that can be larger to current plantation development is carried out fast and effectively distinguishing identification with water cape jasmine, medicinal fructus gardeniae kind.
The method that above-mentioned utilization EST-SSR primers identify cape jasmine kind, wherein, the step one is specifically included：
Total genomic dna is extracted from the blade of discoloration preservation in silica, concrete operation step is as follows：
1) 4ml CTAB extract solutions are added in 10mL centrifuge tubes, 30min is preheated in 65 DEG C of water-baths, 75uL mercaptos are added
Base ethanol；
2) dry 0.5~1.5g of blade is taken, blade master pulse is wiped out and material is placed in clean mortar after shredding, is added suitable
Amount and repeatedly grinds to form fine powdered at liquid nitrogen rapidly, after with spoon it is rapid powder is scraped and goes to fill preheat CTAB extract solutions
10mL centrifuge tubes in, with sealed membrane by centrifuge the mouth of pipe be sealed to prevent that beta -mercaptoethanol from volatilizing, centrifuge tube is then put back into water
30min~45min is incubated in bath, it is frequently light therebetween to shake several times, sample powder and extract solution is fully contacted uniformly, must mix
Liquid；
3) mixed liquor described in taking-up 10ml is placed in centrifuge tube, is placed in mixture of ice and water and is rapidly cooled to room temperature, stands
Isometric 4mL chloroform-isoamyl alcohols are added, the volume ratio of the chloroform-isoamyl alcohol is 24:1, jiggle and be sufficiently mixed
It is even to make it into emulsion, 12000rpm centrifugations 10min under the conditions of then being kept for 4 DEG C, gently by supernatant draw 500uL in
In 1.5ml centrifuge tubes, it is put at once standby in -20 DEG C of refrigerators；
4) the 1.5mL centrifuge tubes for filling 500uL supernatants are taken out, 100uL silica bead suspension is added, uses turbula shaker
About 15s is vibrated, fully mixing is allowed to, in being stored at room temperature after 10min, then vibrates about 5s, 8000rpm centrifuges 15s thereafter, remove
Supernatant produces silica bead-nucleic acid complexes；
5) the centrifuge tube tweezers rule percussion of silica bead-nucleic acid complexes will be filled, makes compound with reference to turbula shaker
Liquid is broken into, is allowed to completely into dispersed, adds GuSCN rinsing liquid 750uL, then the mixing that is fully vortexed with oscillator, after
8000rpm centrifuges 15s, abandons supernatant；
6) step 5 is pressed) repeat rinsing once；
7) the centrifuge tube tweezers rule percussion of silica bead-nucleic acid complexes will be filled, compound is broken into liquid, is added
70% ethanol 1mL, then the mixing that is fully vortexed, the 15s of 8000rpm centrifugations thereafter discard supernatant；
8) step 7 is pressed) repeat rinsing once；
9) compound is broken into liquid with tweezers again, adds absolute ethyl alcohol 1mL, being fully vortexed mixes, afterwards 8000rpm
15s is centrifuged, supernatant is discarded；Then 1.5mL centrifuge tubes lid is opened, is upside down in and is covered with the inclined cardboard of blotting paper, room
Warm air does about 1~2h；
10) addition 150uL TE buffer solutions in the 1.5mL centrifuge tubes of air-dried compound are filled, it is abundant with tweezers rule percussion
After mixing, 10min is incubated in 56 DEG C of water-baths, 12000rpm centrifuges 5min thereafter, it is institute to draw 100uL supernatants with pipette tips
The DNA needed slightly carries solution, and each sample DNA coarse extract for carrying out label is configured into working solution, concentration about 50ng/uL, 4 DEG C of preservations
It is standby；
11) DNA coarse extract passes through UV spectrophotometric determinations yield and purity；And using the inspection of 1% agarose gel electrophoresis
Survey, standard molecular weight is used as using the Marker of Lamda Hind III.
The method that above-mentioned utilization EST-SSR primers identify cape jasmine kind, wherein, the step 2 is specifically included：
14 pairs of SSR primers are used for pcr amplification reaction, all pcr amplification reaction programs are held by PCR amplification instrument
OK, and the optimized EST-SSR mark reaction systems and program for filtering out suitable cape jasmine；
Its reaction system is：Concentration containing 1.5uL is 50ng/uL template DNA, 1uL 10 × Buffer, 0.6uL in 10uL
Concentration is 25mM MgCl2, 0.8uL concentration is 10mM dNTPs, and 0.3uL concentration is 10uM F-primer, 0.3uL concentration
For 10uM R-primer, 0.1uL concentration is 5U/uL Taq enzyme, 5.4Ul ddH2O；
Its response procedures is：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 63 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 15
Individual circulation；94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 15 circulations；72 DEG C of extension 10min；10 DEG C of holdings；
After pcr amplification reaction terminates, sample-loading buffer 6uL is added, fully mixes, is placed in standby in 4 DEG C of refrigerators.
The method that above-mentioned utilization EST-SSR primers identify cape jasmine kind, wherein, the step 3 is specifically included：
Using 8% polyacrylamide gel, it is separated by electrophoresis in Vertial electrophorestic tank, 50bp Marker are used as standard
Molecular weight marker clip size, electrophoresis product carries out data interpretation after silver staining to everybody point, wherein, silver staining step is：It will add
The PCR primer pipette tips of good buffer solution dismantle electrophoresis tank in after 1.5~2h of loading wells loading 0.9uL, 150V constant pressure electrophoresis, will
Short slab is peeled off, and band glue long slab is used for fixed dye；Fixed 10min, wherein fixer are 10% ethanol, 0.5% pure acetic acid,
ddH2O is rinsed 2 times, each 1min；0.15%AgNO is used again3Dye 8~15min, ddH2O is rinsed 2 times, each 2min；Then will
Band glue glass plate, which is put into developer solution, to develop untill amplified band is clear, fixed, rinsing, the progress on shaking table of developing, will
Glass plate is cleaned twice with running water, is placed on film illuminator, finally uses digital photographing Taking Pictures recording.
The method that above-mentioned utilization EST-SSR primers identify cape jasmine kind, wherein, the step 4 is specifically included：
EST-SSR amplifications are carried out to different cape jasmine kinds respectively with 14 pairs of EST-SSR primers, different cape jasmine product are obtained
The 14 EST-SSR amplified productions planted；Electrophoresis detection difference cape jasmine 14 EST-SSR amplified productions of kind, if different cape jasmine product
At least five amplification banding pattern is different in 14 EST-SSR amplified productions of two cape jasmine kinds of certain in kind, then two cape jasmine kinds
For different cultivars；If being less than 5 amplified bands in 14 EST-SSR amplified productions of certain two cape jasmine kind in different cape jasmine kinds
Type is different, then two cape jasmine kinds are same breed.
The method that above-mentioned utilization EST-SSR primers identify cape jasmine kind, wherein, the step 4 is specifically included：
EST-SSR amplifications are carried out to different cape jasmine kinds respectively with 14 pairs of EST-SSR primer pairs, different cape jasmines are obtained
14 EST-SSR amplified productions of kind；Different cape jasmine 14 EST-SSR amplified productions of kind described in electrophoresis detection, will be described
14 EST-SSR amplified productions of different cape jasmine kinds represent that bar is carried with " 1 " respectively, and " 0 " represents band without structure 14
Molecular marking fingerprint, each molecular marking fingerprint 1 EST-SSR amplified production of correspondence and 1 primer pair；If different
At least five is different in 14 molecular fingerprint collection of illustrative plates of certain two cape jasmine kind in cape jasmine kind, then two cape jasmine kinds is not
Same kind；If being less than 5 differences, this two in 14 molecular fingerprint collection of illustrative plates of certain two cape jasmine kind in different cape jasmine kinds
Cape jasmine kind is same breed.
Another aspect of the present invention also provides a kind of application of the above method, is expanded by 14 pairs of EST-SSR primer PCRs
EST-SSR spectral band analysis are carried out after increasing, to carrying out cultivar identification with known cape jasmine kind for examination cape jasmine kind.
Another aspect of the present invention also provides a kind of application of the above method, is expanded by 14 pairs of EST-SSR primer PCRs
EST-SSR spectral band analysis are carried out after increasing, cape jasmine Variety fingerprinting is built.
Brief description of the drawings
Fig. 1 is that EST-SSR primers eGJ019 expands electrophoresis detection structure chart to the PCR of 1-16 parts of samples before cape jasmine kind；
Fig. 2 is that EST-SSR primers eGJ019 expands electrophoresis detection structure chart to the PCR of 17-39 parts of samples after cape jasmine kind；
Fig. 3 is that EST-SSR primers eGJ026 expands electrophoresis detection structure chart to the PCR of 1-16 parts of samples before cape jasmine kind；
Fig. 4 is that EST-SSR primers eGJ026 expands electrophoresis detection structure chart to the PCR of 17-39 parts of samples after cape jasmine kind；
Fig. 5 is that EST-SSR primers eGJ134 expands electrophoresis detection structure chart to the PCR of 1-16 parts of samples before cape jasmine kind；
Fig. 6 is that EST-SSR primers eGJ134 expands electrophoresis detection structure chart to the PCR of 17-39 parts of samples after cape jasmine kind；
Fig. 7 is the affiliation dendrogram of 39 parts of cape jasmine sample rooms.
Embodiment
For the ease of understanding the present invention, the present invention is described more fully below with reference to relevant drawings.In accompanying drawing
Give some embodiments of the present invention.But, the present invention can be realized in many different forms, however it is not limited to this paper institutes
The embodiment of description.On the contrary, the purpose that these embodiments are provided be make to the disclosure more it is thorough comprehensively.
Unless otherwise defined, all of technologies and scientific terms used here by the article is with belonging to technical field of the invention
The implication that technical staff is generally understood that is identical.Term used in the description of the invention herein is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein " and/or " include one or more phases
The arbitrary and all combination of the Listed Items of pass.
In the present embodiment, 39 parts of cape jasmine kind material is chosen first, and cape jasmine kind sampling material is specific such as table 1, Mei Gepin
1~5 part of sample is planted, sampling method, sample preservation use prior art, will not be described here.
The cape jasmine kind of table 1 samples material
Step one, the DNA of experimental cultivar sample is extracted：
In the present embodiment, extract total from the blade of discoloration preservation in silica using improved CTAB cracking-silica bead absorption method
Genomic DNA, concrete operation step is as follows：
1) added in 10ml centrifuge tubes 4ml CTAB extract solutions (CTAB concentration is 0.1g/L, and PVP concentration is 0.5g/L,
NaCl 1.4mol/L, EDTA 20mmol/L (pH8.0), Tris-HCl 100mmol/L (pH8.0)), in 65 DEG C of water-baths
30min is preheated, 75uL mercaptoethanols are added；
2) dry 2~4, blade (about 0.5~1.5g) is taken, remaining blade material is put back to standby in valve bag, wipes out blade
Material is simultaneously placed in clean mortar after shredding by master pulse, is added appropriate liquid nitrogen and is repeatedly ground to form fine powdered, rear medication rapidly
Spoon is rapid to be scraped powder and goes in the 10mL centrifuge tubes for filling preheating CTAB extract solutions, is sealed with sealed membrane by the mouth of pipe is centrifuged
To prevent that beta -mercaptoethanol from volatilizing, centrifuge tube is then put back into insulation 30min~45min in water-bath, gently shaken frequently therebetween several
It is secondary, sample powder and extract solution is fully contacted uniformly, obtain mixed liquor；
3) mixed liquor described in taking-up 10mL is placed in centrifuge tube, is placed in mixture of ice and water and is rapidly cooled to room temperature, stands
Add isometric 4mL chloroform-isoamyl alcohols (24:1, V/V), jiggle to be sufficiently mixed and uniformly make it into emulsion, then
12000rpm centrifugations 10min under the conditions of being kept for 4 DEG C, gently draws 500uL in 1.5mL centrifuge tubes by supernatant, be put at once-
It is standby in 20 DEG C of refrigerators；
4) the 1.5mL centrifuge tubes for filling 500uL supernatants are taken out, 100uL silica beads (SiO is added2) suspension, use vortex
Oscillator vibrates about 15s, is allowed to fully mixing, in being stored at room temperature after 10min, then vibrates about 5s, 8000rpm is centrifuged thereafter
15s, removes supernatant and produces silica bead-nucleic acid complexes；
5) the centrifuge tube tweezers rule percussion of silica bead-nucleic acid complexes will be filled, makes compound with reference to turbula shaker
Liquid is broken into, is allowed to completely into dispersed, adds GuSCN rinsing liquid 750ul, then the mixing that is fully vortexed with oscillator, after
8000rpm centrifuges 15s, abandons supernatant；
6) step 5 is pressed) repeat rinsing once；
7) the centrifuge tube tweezers rule percussion of silica bead-nucleic acid complexes will be filled, compound is broken into liquid, is added
70% ethanol 1mL, then the mixing that is fully vortexed, the 15s of 8000rpm centrifugations thereafter discard supernatant；
8) step 7 is pressed) repeat rinsing once；
9) compound is broken into liquid with tweezers again, adds absolute ethyl alcohol 1mL, being fully vortexed mixes, thereafter 8000rpm
15s is centrifuged, supernatant is discarded；Then 1.5mL centrifuge tubes lid is opened, is upside down in and is covered with the inclined cardboard of blotting paper, room
Warm air does about 1~2h；
10) addition 150Ul TE buffer solutions in the 1.5mL centrifuge tubes of air-dried compound are filled, it is abundant with tweezers rule percussion
After mixing, 10min is incubated in 56 DEG C of water-baths, 12000rpm centrifuges 5min thereafter.It is institute to draw 100uL supernatants with pipette tips
The DNA needed slightly carries solution.The each sample DNA coarse extract for carrying out label is configured to working solution, concentration about 50ng/ μ L, 4 DEG C of preservations
It is standby；
11) DNA coarse extract is determined by UV spectrophotometers (ND-1000, Thermo Electron Corporation)
Yield and purity；And detected using 1% agarose gel electrophoresis, the Marker of Lamda Hind III (Mbi Fermentas,
Shenzhen, Guangdong, China) it is used as standard molecular weight.
Step 2, pcr amplification reaction is carried out using EST-SSR primers, and the EST-SSR primers are 14 pairs, and its sequence is such as
Under：
2 14 pairs of EST-SSR primers of table
All pcr amplification reaction programs by the grads PCR amplification instruments of Eppendorf 5331 (Eppendorf, Hamburg,
Germany) perform, and the optimized EST-SSR mark reaction systems and program for filtering out suitable cape jasmine.Its reaction system is：
Template DNA containing 1.5uL (50ng/uL) in 10uL, 10 × Buffer of 1uL (Mg2+Free), 0.6uL MgCl2(25mM), 0.8uL
DNTPs (10mM), 0.3uL F-primer (10uM), 0.3uL R-primer (10uM), 0.1uL Taq enzymes (5U/uL)
(TaKaRa Bio Inc., Otsu, Shiga, Japan), 5.4uL ddH2O.Response procedures are：94 DEG C of pre-degeneration 3min；94℃
It is denatured 30s, 63 DEG C of annealing 30s (1 DEG C of often circulation reduction), 72 DEG C of extension 30s, totally 15 circulations；94 DEG C of denaturation 30s, 57 DEG C are moved back
Fiery 30s, 72 DEG C of extension 30s, totally 15 circulations；72 DEG C of extension 10min；10 DEG C of holdings.After pcr amplification reaction terminates, in addition
Sample buffer solution (0.25% bromjophenol blue, 15% ficoll) 6uL, fully mixes, is placed in standby in 4 DEG C of refrigerators.
Step 3, Polyacrylamide Gel Electrophoresis：
Using the 8% polyacrylamide gel (glue (acrylamide containing 10ml 40% in 50mL encapsulating liquid:Methene acrylamide
=39:1 (w/w), urea 21g, 6.25mL 10 × TBE, 350 μ L APS, 50 μ L TEMED), electricity is carried out in Vertial electrophorestic tank
Swimming separation, 50bp Marker (TIANGEN, Beijing, China) are used as Standard molecular weight markers clip size, electrophoresis product
Data interpretation is carried out to everybody point after silver staining.Silver staining step is：The PCR primer pipette tips of buffer solution will have been added in loading wells
After the 1.5~2h of μ L, 150V constant pressure electrophoresis of sample 0.9, electrophoresis tank is dismantled, short slab is peeled off, band glue long slab is used for fixed dye；It is fixed
10min (ethanol of fixer 10%, 0.5% pure acetic acid (v/v)), ddH2O are rinsed 2 times, each 1min；0.15%AgNO3 is used again
(w/v) 8~15min is dyed, ddH2O is rinsed 2 times, each 2min；Then it will be put into glue glass plate in developer solution and (match somebody with somebody 100mL
1.5gNaOH, 1mL 0.756% sodium tetraborate, 0.75mL formaldehyde need to be added) develop untill amplified band is clear.Fixed, drift
Wash, develop and carried out on shaking table.Glass plate is cleaned twice with running water, is placed on film illuminator, finally with digital photograph
Phase Taking Pictures recording.Primer eGJ19, eGJ26, eGJ134 are shown in Fig. 1 to the PCR amplifications electrophoresis detection result of 39 parts of kind samples of cape jasmine
To Fig. 6.
Step 4, EST-SSR spectral band analysis：
EST-SSR belongs to codominant marker, if the amplified production of same primer pair different templates is in electrophoretic mobility one
Cause, that is, be considered as same site, i.e., with homology.Many people's interpretation alignments are used to glue diagram data to the picture of gained
Interpretation is carried out, is numbered from big to small with A, B, C, D, E ... by band length size, i.e., uppermost allele band is
A, tape reading result such as AB, BC etc., if some sample only has a band, tape reading result is then AA, BB etc..Count everybody simultaneously
The clip size of point band, facilitates value data to change, and strip analysis will according to software format all results after finishing
The data processing and statistical analysis for asking input corresponding software to be tested.
EST-SSR amplifications are carried out to different cape jasmine kinds respectively with 14 pairs of EST-SSR primers, different cape jasmine product are obtained
The 14 EST-SSR amplified productions planted；Electrophoresis detection difference cape jasmine 14 EST-SSR amplified productions of kind, if different cape jasmine product
At least five amplification banding pattern is different in 14 EST-SSR amplified productions of two cape jasmine kinds of certain in kind, then two cape jasmine kinds
For different cultivars；If being less than 5 amplified bands in 14 EST-SSR amplified productions of certain two cape jasmine kind in different cape jasmine kinds
Type is different, then two cape jasmine kinds are same breed.
Further, it is also possible to using analysis below method：
EST-SSR amplifications are carried out to different cape jasmine kinds respectively with 14 pairs of EST-SSR primer pairs, different cape jasmines are obtained
14 EST-SSR amplified productions of kind；Different cape jasmine 14 EST-SSR amplified productions of kind described in electrophoresis detection, will be described
14 EST-SSR amplified productions of different cape jasmine kinds represent that bar is carried with " 1 " respectively, and " 0 " represents band without structure 14
Molecular marking fingerprint, each molecular marking fingerprint 1 EST-SSR amplified production of correspondence and 1 primer pair；If different
At least five is different in 14 molecular fingerprint collection of illustrative plates of certain two cape jasmine kind in cape jasmine kind, then two cape jasmine kinds is not
Same kind；If being less than 5 differences, this two in 14 molecular fingerprint collection of illustrative plates of certain two cape jasmine kind in different cape jasmine kinds
Cape jasmine kind is same breed.
In the present embodiment, 14 pairs of EST-SSR primers have obtained preferable polymorphic in for examination cape jasmine kind sample
Amplification.10 cape jasmine kinds and 1 cape jasmine cultivate original seed totally 39 parts of materials, using genetic distance matrix as analysis object, utilize
The softwares of NTSYS-pc 2.1 carry out cluster analysis by matched pair technique (UPGMA) in groups is not weighted, referring to Fig. 7, establishing 10 Cape jasmines
The affiliation of sub- kind and 1 cape jasmine cultivation original seed totally 39 parts of storerooms is tree-shaped.From the point of view of cluster result, except cultivation cape jasmine is
5 samples of fructus gardeniae, golden 3 samples of good fortune water Cape jasmine and great Hua cape jasmines do not flock together completely, especially golden good fortune water 3 samples of Cape jasmine
Cluster is more disperseed, and other each sample homopolymerizations of 8 kinds gather together.Wherein wide rib water Cape jasmine is shown and other kind affiliations
Farthest, lotus cape jasmine shows and Taihu Lake mountain Cape jasmine, white toad affiliation are nearer, cone cape jasmine show with fructus gardeniae affiliation compared with
Closely, small white toad is shown and water cape jasmine affiliation is nearer, and great Hua cape jasmines, the cluster display of tongue of sparrow cape jasmine are then more independent.
The embodiment provides the application of the above method, expanded by 14 pairs of EST-SSR primer PCRs laggard
Row EST-SSR spectral band analysis, to carrying out cultivar identification with known cape jasmine kind for examination cape jasmine kind.
In addition, embodiments of the invention additionally provide the application of the above method, draw specifically by 14 couples of EST-SSR
EST-SSR spectral band analysis are carried out after thing PCR amplifications, cape jasmine EST-SSR finger-prints are built.
The electrophoresis result expanded according to polymorphic primer, has band to be designated as " 1 ", no band is designated as using on identical mobility position
The tape reading method of " 0 ", the finger-print of 10 kinds of cape jasmine is built with 14 EST-SSR marks, table 3 is for details, reference can be made to, can by 3
See, primer eGJ15 and eGJ19 polymorphism are enriched the most, they combine and 10 cape jasmine kinds can distinguished substantially together, its
He can also complete the differentiation to all kinds of cape jasmine by primer together by 3 or 3 combination of the above.
The finger-print of 3 10, table cape jasmine kind 14 EST-SSR marks
The method that utilization EST-SSR primers that the present invention is provided identify cape jasmine kind, cape jasmine kind can be carried out it is quick,
Effectively identify, and qualification result is difficult to be influenceed by plant age, can accurately distinguish Seedling Stage cape jasmine kind, can be to kind at present
Hair transplant opens up larger pigment and is carried out fast and effectively distinguishing identification with water cape jasmine, medicinal fructus gardeniae kind.Can look forward to plantation
Industry, peasant household avoid the heavy losses by the wrong kind of plantation, and the cape jasmine EST-SSR finger-prints set up based on the above method
Distinguishing significant cape jasmine kind.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (8)
1. a kind of method that utilization EST-SSR primers identify cape jasmine kind, it is characterised in that including：
Step one, the DNA of experimental cultivar sample is extracted；
Step 2, pcr amplification reaction is carried out using EST-SSR primers, and the EST-SSR primers are 14 pairs, and its sequence is as follows：
Step 3, Polyacrylamide Gel Electrophoresis；
Step 4, EST-SSR spectral band analysis build molecular marking fingerprint.
2. the method that utilization EST-SSR primers according to claim 1 identify cape jasmine kind, it is characterised in that the step
Rapid one specifically includes：
Total genomic dna is extracted from the blade of discoloration preservation in silica, concrete operation step is as follows：
1) 4ml CTAB extract solutions are added in 10mL centrifuge tubes, 30min is preheated in 65 DEG C of water-baths, 75uL sulfydryl second is added
Alcohol；
2) dry 0.5~1.5g of blade is taken, blade master pulse is wiped out and material is placed in clean mortar after shredding, appropriate liquid is added
Nitrogen simultaneously repeatedly grinds to form fine powdered rapidly, after with spoon it is rapid powder is scraped and gone to fill preheating CTAB extract solutions
In 10mL centrifuge tubes, it is sealed with sealed membrane by the mouth of pipe is centrifuged to prevent that beta -mercaptoethanol from volatilizing, centrifuge tube is then put back into water-bath
30min~45min is incubated in pot, it is frequently light therebetween to shake several times, sample powder and extract solution is fully contacted uniformly, must mix
Liquid；
3) mixed liquor described in taking-up 10ml is placed in centrifuge tube, is placed in mixture of ice and water and is rapidly cooled to room temperature, adds immediately
Enter isometric 4mL chloroform-isoamyl alcohols, the volume ratio of the chloroform-isoamyl alcohol is 24:1, jiggling to be sufficiently mixed uniformly makes
It is into emulsion, 12000rpm centrifugations 10min under the conditions of then being kept for 4 DEG C, gently by supernatant draw 500uL in 1.5ml from
In heart pipe, it is put at once standby in -20 DEG C of refrigerators；
4) the 1.5mL centrifuge tubes for filling 500uL supernatants are taken out, 100uL silica bead suspension is added, is vibrated using turbula shaker
About 15s, is allowed to fully mixing, in being stored at room temperature after 10min, then vibrates about 5s, 8000rpm centrifuges 15s thereafter, remove supernatant
Liquid produces silica bead-nucleic acid complexes；
5) the centrifuge tube tweezers rule percussion of silica bead-nucleic acid complexes will be filled, compound is broken into reference to turbula shaker
Liquid, is allowed to completely into dispersed, adds GuSCN rinsing liquid 750uL, then be fully vortexed with oscillator mixing, rear 8000rpm
15s is centrifuged, supernatant is abandoned；
6) step 5 is pressed) repeat rinsing once；
7) the centrifuge tube tweezers rule percussion of silica bead-nucleic acid complexes will be filled, compound is broken into liquid, adds 70%
Ethanol 1mL, then the mixing that is fully vortexed, the 15s of 8000rpm centrifugations thereafter discard supernatant；
8) step 7 is pressed) repeat rinsing once；
9) compound is broken into liquid with tweezers again, adds absolute ethyl alcohol 1mL, being fully vortexed is mixed, and 8000rpm is centrifuged afterwards
15s, discards supernatant；Then 1.5mL centrifuge tubes lid is opened, is upside down in and is covered with the inclined cardboard of blotting paper, room temperature wind
Dry about 1~2h；
10) addition 150uL TE buffer solutions in the 1.5mL centrifuge tubes of air-dried compound are filled, are fully mixed with the percussion of tweezers rule
Afterwards, 10min is incubated in 56 DEG C of water-baths, 12000rpm centrifuges 5min thereafter, is drawn with pipette tips needed for 100uL supernatants are
DNA slightly carries solution, and each sample DNA coarse extract for carrying out label is configured into working solution, and concentration about 50ng/uL, 4 DEG C of preservations are standby
With；
11) DNA coarse extract passes through UV spectrophotometric determinations yield and purity；And using the detection of 1% agarose gel electrophoresis, adopt
Standard molecular weight is used as with the Marker of Lamda Hind III.
3. the method that utilization EST-SSR primers according to claim 1 identify cape jasmine kind, it is characterised in that the step
Rapid two specifically include：
14 pairs of EST-SSR primers are used for pcr amplification reaction, all pcr amplification reaction programs are held by PCR amplification instrument
OK, and the optimized EST-SSR mark reaction systems and program for filtering out suitable cape jasmine；
Its reaction system is：Concentration containing 1.5uL is 50ng/uL template DNA, 1uL 10 × Buffer, 0.6uL concentration in 10uL
For 25mM MgCl2, 0.8uL concentration is 10mM dNTPs, and 0.3uL concentration is 10uM F-primer, and 0.3uL concentration is
10uM R-primer, 0.1uL concentration are 5U/uL Taq enzyme, 5.4Ul ddH2O；
Its response procedures is：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 63 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 15 are followed
Ring；94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 15 circulations；72 DEG C of extension 10min；10 DEG C of holdings；PCR
After amplified reaction terminates, sample-loading buffer 6uL is added, fully mixes, is placed in standby in 4 DEG C of refrigerators.
4. the method that utilization EST-SSR primers according to claim 1 identify cape jasmine kind, it is characterised in that the step
Rapid three specifically include：
Using 8% polyacrylamide gel, it is separated by electrophoresis in Vertial electrophorestic tank, 50bp Marker are used as standard molecule
Labeled fragment size is measured, electrophoresis product carries out data interpretation after silver staining to everybody point, wherein, silver staining step is：Delay adding
The PCR primer pipette tips of fliud flushing dismantle electrophoresis tank, by short slab in after 1.5~2h of loading wells loading 0.9uL, 150V constant pressure electrophoresis
Peel off, band glue long slab is used for fixed dye；Fixed 10min, wherein fixer are 10% ethanol, 0.5% pure acetic acid, ddH2O floats
Wash 2 times, each 1min；0.15%AgNO is used again3Dye 8~15min, ddH2O is rinsed 2 times, each 2min；Then will be with glue glass
Glass plate, which is put into developer solution, develops untill amplified band is clear, fixed, rinsing, the progress on shaking table of developing, by glass plate
Cleaned twice, be placed on film illuminator with running water, finally use digital photographing Taking Pictures recording.
5. the method that utilization EST-SSR primers according to claim 1 identify cape jasmine kind, it is characterised in that the step
Rapid four specifically include：
EST-SSR amplifications are carried out to different cape jasmine kinds respectively with 14 pairs of EST-SSR primers, different cape jasmine kinds are obtained
14 EST-SSR amplified productions；Electrophoresis detection difference cape jasmine 14 EST-SSR amplified productions of kind, if in different cape jasmine kinds
At least five amplification banding pattern is different in 14 EST-SSR amplified productions of certain two cape jasmine kind, then two cape jasmine kinds is not
Same kind；If being less than 5 amplification banding patterns in 14 EST-SSR amplified productions of certain two cape jasmine kind not in different cape jasmine kinds
Together, then two cape jasmine kinds are same breed.
6. the method that utilization EST-SSR primers according to claim 1 identify cape jasmine kind, it is characterised in that the step
Rapid four specifically include：
EST-SSR amplifications are carried out to different cape jasmine kinds respectively with 14 pairs of EST-SSR primer pairs, different cape jasmine kinds are obtained
14 EST-SSR amplified productions；Different cape jasmine 14 EST-SSR amplified productions of kind described in electrophoresis detection, by the difference
14 EST-SSR amplified productions of cape jasmine kind represent that bar is carried with " 1 " respectively, and " 0 " represents band without 14 molecules of structure
Marking fingerprint, each molecular marking fingerprint 1 EST-SSR amplified production of correspondence and 1 primer pair；If different cape jasmines
At least five is different in 14 molecular fingerprint collection of illustrative plates of two cape jasmine kinds of certain in kind, then two cape jasmine kinds are different product
Kind；If being less than 5 differences, two cape jasmines in 14 molecular fingerprint collection of illustrative plates of certain two cape jasmine kind in different cape jasmine kinds
Kind is same breed.
7. a kind of application of claim 1 to 6 any one methods described, it is characterised in that drawn by 14 couples of EST-SSR
EST-SSR spectral band analysis are carried out after thing PCR amplifications, to carrying out cultivar identification with known cape jasmine kind for examination cape jasmine kind.
8. a kind of application of claim 1 to 6 any one methods described, it is characterised in that drawn by 14 couples of EST-SSR
EST-SSR spectral band analysis are carried out after thing PCR amplifications, cape jasmine Variety fingerprinting is built.
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