CN108531545A - A method of screening fist rolls up marchantia SSR primers - Google Patents
A method of screening fist rolls up marchantia SSR primers Download PDFInfo
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Abstract
The present invention discloses a kind of method of screening fist volume marchantia SSR primers, using SAM methods, to wild species fist volume marchantia (Marchantia convoluta) progress SSR molecular marker research.DNA is extracted using the CTAB methods of improvement, then marchantia SSR primers are rolled up through digestion, connection, inhibition PCR, pre- amplification, SAM methods, PCR screening fist.SAM method amplification bands are clear, and screening obtains positive colony and is sequenced, and obtains SSR primer sequences.The fist volume marchantia SSR primers effectively expanded are filtered out by PCR.The SSR molecular marker technology can be used as fist volume marchantia genetic diversity research method.
Description
Technical field
The invention belongs to genetic molecule technical fields, are related to natural resources of Chinese medicinal materials utilization and genetic diversity Journal of Sex Research, specifically
It is related to a kind of method of screening fist volume marchantia SSR primers.
Background technology
Currently, carrying out genetics research to organism with molecular labeling has become emphasis and hot spot under birth, how
Select proper method marginal to the efficiency of genetic research and success or failure tool in numerous molecular labelings.And in numerous molecule marks
In note technology, expanding based on SSR marker specificity, good stability and repeatability, the heredity that can preferably reflect species
The features such as structure and genetic diversity change, is considered as more satisfactory genetic marking method.Especially in recent years cultivar identification,
Geographic origin differentiates to be applied in research, population genetic research, active ingredient and genetic diversity research etc..However SSR is marked
Note has species specificity, and the SSR primers between different plant species do not have versatility, it is necessary to for each species develop special primer into
Row PCR detections.
It is that Marchantiaceae marchantia fist rolls up marchantia that fist, which rolls up marchantia through Guangxi University of Chinese Medicine's pharmacognosy expert appraisal,
(Marchantia convoluta Gao et Chang), picks up from Guangxi various regions.According to the literature, marchantia crude drug is rolled up about fist
It learns and differentiates research, analysis of volatile components, flavone compound research, liposoluble constituent research, anti-acute liver damage and anti-second
Hepatovirus pharmacological research and the research of ISSR-PCR amplification systems appear in the newspapers, but have no at present about in SSR genetic marker researchs
Hold.Marchantia DNA is rolled up using the CTAB methods extraction fist of improvement herein, is expanded through digestion, connection, inhibition PCR, pre- amplification, SAM methods
Expected segment is obtained, the fist volume marchantia SSR primers effectively expanded are filtered out by PCR, marchantia molecule resource crude drug mirror is rolled up for fist
Not, resources development and utilization provides technical foundation.
Invention content
The object of the present invention is to provide a kind of methods that screening fist rolls up marchantia SSR primers, and thus method obtains fist volume
Marchantia SSR primers, by rolling up the research of marchantia SSR primer screenings to fist, it is right to have obtained fist volume marchantia SSR primers 30, is a kind of
It studies fist and rolls up the preferable research method of marchantia genetic diversity.Marchantia DNA is rolled up using the CTAB methods extraction fist of improvement, through digestion, even
Connect, inhibition PCR, pre- amplification, SAM methods expand to obtain expected segment, the fist volume marchantia SSR effectively expanded is filtered out by PCR
Primer rolls up the discriminating of marchantia molecule resource crude drug for fist, resources development and utilization provides technical foundation.
Technical scheme is as follows：
A method of screening fist rolls up marchantia SSR primers, and this method extracts the genomic DNA of fist volume marchantia first, then
Carry out digestion and connection, inhibition PCR, expanded in advance again and SAM methods amplification, PCR screening punch volume marchantia SSR primers.
As being further improved for technical solution, a kind of above-described method of screening fist volume marchantia SSR primers is special
Sign is：The genomic DNA of the extraction fist volume marchantia is to roll up to extract fist volume ground in marchantia blade from fist using CTAB methods
The genome DNA sample of money.Ultraviolet specrophotometer measures the genome of the fist volume marchantia of the CTAB methods extraction of display improvement
The OD 260/OD280 ratios of DNA, while detecting DNA purity through 1% agarose gel electrophoresis.
As being further improved for technical solution, a kind of above-described method of screening fist volume marchantia SSR primers is described
Digestion, be connected as with restriction enzyme Mse I and Pst I to fist roll up marchantia genome DNA sample carry out double digestion and
Connection obtains fist volume marchantia DNA enzymatic and cuts and connection product, and fist volume marchantia DNA enzymatic is cut and 1% Ago-Gel of connection product electricity
Swimming detection, clip size 100-1500bp.
As being further improved for technical solution, a kind of above-described method of screening fist volume marchantia SSR primers is described
Pre-expansion increase to fist volume marchantia DNA enzymatic cut and expanded in advance with connection product, obtain pre- amplified production, the pre- amplification item
Part is： Takara Ex Taq 1U；10×Ex Taq Buffer 2μl；The 1 μ l of suppression PCR products diluted；
25mMMgC12 1.5μl；1.5 μ l of dNTP Mix (each 2.5mM)；Mse I adapter Primer 1 5pmol；Pst I
adapter primer 5pmol；DdH20 to 20 μ l；Wherein：
The sequence of Mse I adapter primer 1：5'-GAC GAC CGA CGA GTA AC-'3
The sequence of Pst I adapter primer：5'-AGA CTG CGT ACA TGC AGGA-'3.
As being further improved for technical solution, a kind of above-described method of screening fist volume marchantia SSR primers is described
SAM methods amplification be to be in reaction system by pre- amplified production：10×Ex Taq Buffer 2μl；The pre-expansion volume increase diluted
2 μ l of object； Takara Ex Taq lU；25mM MgCl2 1.5μl；1.5 μ l of dNTP Mix (each 2.5mM)；Mse I adapter
Primer 3 (4~10) 5pmol；SSR primer(PCT6)20pmol；Add ddH20 to 20 μ l, under conditions of carry out SAM expansions
Increase, obtains SAM amplified productions.
As being further improved for technical solution, a kind of above-described method of screening fist volume marchantia SSR primers is described
The amplification of SAM methods further include SAM amplified productions recycling and clone, the recycling of the SAM amplified productions is with clone：It is logical
It crosses native polyacrylamide gel electrophoresis to detach SAM amplified productions, selects 200 SAM amplification pieces of different sizes at random
Section, pipette tips crushing simultaneously impregnate centrifugation with distilled water, carry out second of SAM amplification, and amplified production is purified with agarose gel electrophoresis,
DNA purification kits recycle, and recovery product is transferred to competent cell, through Amp+ by the connection with PMD 18-T Vector
LB plate screenings obtain positive colony, select 2 white colonies from each tablet at random, and clone has obtained 100 SAM pieces altogether
Section is for being sequenced.
As being further improved for technical solution, a kind of above-described method of screening fist volume marchantia SSR primers is described
PCR screening be：The flanking sequence of SSR contained in the DNA fragmentation obtained according to sequencing uses Primer premier 5.0
Software for Design special primer is synthesized from 86 segments according to the one or both sides sequence design special primer in the sites SSR,
Then primer pair is formed with 5 ' anchoring degenerate primer SSR primer (PCT6), using the genomic DNA of fist volume marchantia as template,
It is detected through 2% agarose gel electrophoresis, filters out the special primer of 30 pairs of clear bands, as fist rolls up marchantia SSR primers.
As being further improved for technical solution, a kind of above-described method of screening fist volume marchantia SSR primers is described
PCR screening be：Further include being measured to show with ultraviolet specrophotometer after the genome DNA sample for getting fist volume marchantia
Show the OD 260/OD280 ratios for the genomic DNA for rolling up marchantia with fist, while DNA purity is detected through 1% agarose gel electrophoresis
Beneficial effects of the present invention are：
1. the present invention devises the SSR primers that fist rolls up marchantia using SAM methods for the first time；
2. the present invention utilizes the CTAB methods extraction fist volume marchantia DNA of improvement；
3. the present invention expands to obtain expected segment, ligation amplification production through digestion, connection, inhibition PCR, pre- amplification, SAM methods
Object converts Escherichia coli, random picking hickie, and screening obtains 100 positive colonies and is sequenced, and obtains 86 sequences of SSR primers
Row.It is right that the fist volume marchantia SSR primers 30 effectively expanded are filtered out by PCR；
4. SSR molecular marker of the present invention will roll up the cultivar identification of marchantia, genetic analysis, genetic map applied to fist
It establishes, the every field such as sort research and germplasm identification.
Description of the drawings
Fig. 1 SAM amplified production electrophoretograms
Fig. 2 primer electrophoretograms
Specific implementation mode
1 test method and step：
(1) extraction and purification fist rolls up marchantia genomic DNA
800 μ l CTAB extracts are drawn in 2ml centrifuge tubes, 60 DEG C of water-bath preheatings；Addition 0.1g fists volume marchantia blade, 200
μ l CTAB extracts are transferred to after being fully ground in step (1) centrifuge tube；60 DEG C are incubated 2 hours, and 15min shakes up once；Room temperature
8000rpm, centrifuges 6min, and Aspirate supernatant is transferred to 2ml centrifuge tubes；Isometric phenol is added：Chloroform：Isoamyl alcohol (25:24:1),
It is sufficiently mixed；8000rpm centrifuges 10min, recycles water phase；Isometric chloroform-isoamyl alcohol (24 is added:1) it, then extracts primary；
Absorb water phase, adds 1/10 times of volume NaAC (5.2M), 2.5 times of frost absolute ethyl alcohols, and 8000rpm after mixing centrifuges 5min；It is heavy to collect
It forms sediment, 75% ethyl alcohol washs 3 times, air-dries；Sediment deionized water dissolving；RNAase liquid 3 μ l, 35 DEG C of water-bath 20min is added；
After water-bath, isometric phenol is added:Chloroform:Isoamyl alcohol (25:24:L) it extracts again；10000rpm centrifuges 5min, and water suction phase turns
New centrifuge tube；Centrifuge tube ice bath, the NaAc of 1/10 volume, 2 times of volumes frost absolute ethyl alcohol be added in centrifuge tube, -20 DEG C of ice
Case is stayed overnight；4 DEG C, 12000rpm centrifuges 5min, collects precipitation；75% ethyl alcohol is washed 2 times, is air-dried；The deionized water dissolving of 100 μ l
DNA, -20 DEG C save backup.
(2) digestions of fist volume marchantia DNA sample and detection
The reaction system of digestion with restriction enzyme：10 μ l of genome DNA sample；10×NEB buffer II 5.0μl；
Mse I 5U；Pst I 5U；Deionized water is mended to 50 μ l of total volume.Above-mentioned condition keeps the temperature 2h in 36 DEG C, and subsequent 65 DEG C of water-baths are protected
Warm 10min, stops endonuclease reaction, and 2% Ago-Gel detects digestion effect, -20 DEG C of preservations of resultant product.
(3) connection of the digestion and connector of fist volume marchantia genome DNA sample
Following procedure is carried out after digestion effect qualification：
Mse I and Pst I connectors are annealed
Annealing buffer dissolving Mse I adapter Sense strand and Mse I adapter are used respectively
antisense strand；It draws equimolar above two solution and cryopreservation tube mixing is added, 5000rpm centrifuges 10S；65 DEG C of water-baths
10min is kept the temperature, it is cooling.Pst I connector method for annealing is same as above.
It is attached：annealing buffer:10mM Tris·cl,pH7.5
100mM NaCi
1mM EDTA, pH8.0
The sequence of Mse I adapter sense strand:5'-GAG CAA GGC TCT CAC AAG GAC GAC
CGA CGA G-'3
The sequence of Mse I adapter antisense strand:5'phos-TAC TCG TCG GTC GTC CTT
GTG AGA GCC TTG CT-'3
The sequence of Pst I adapter sense strand:5'-CTC GGA AGC CTC AGT CCC AGA CTG
CGT ACA TGC A-'3
The sequence of Pst I adapter antisense strand:5'phos-TGT ACG CAG TCT GGC ACT
GCT TCC GAG A-'3
The restricted double digestion of DNA sample
Digestion system：20 μ l of genome DNA sample；Mse I 0.5μl；PSt I 0.5μl；10×NEB buffer II
5μl； BSA 0.5μl；ddH2O 50μl.Mixing, centrifugation, is put into 37 DEG C of water-bath 2h of water bath.
DNA enzymatic cuts sample jointing
It draws 50pmol Mse I and 5pmol Pst I connectors to be added in DNA, 45 DEG C of water-bath 5min.Then it is added：
10mg/ml BSA 0.05μl、10×NEB buffer II 0.5μl、T4 1igase 0.5μl、10mM dATP 3.5μl、
DdH20 to 60 μ l；5000rpm centrifuges 10S；37 DEG C of heat preservation 1h, are placed at room temperature for 2h.
(4) Suppression PCR and detection
Reaction system：10×EX Taq Buffer 2μl；25mM MgC12 1.5μl；Oneself completes the DNA of digestion and connection
4 μ l of sample；1.5 μ l of dNTP Mix (each 2.5mM)；Takara ExTaq lU；Mse I suppressor primer 5pmol；
Pst I suppressor primer 5pmol；DdH2O to 20 μ l.5000rpm centrifuges 10S.PCR expansions are carried out by the following conditions
Increase：94 DEG C of pre-degeneration 1min；It is denaturalized 94 DEG C of 1min, 56 DEG C of renaturation 1min, 72 DEG C of extension 1min, after 25 recycle；72 DEG C are prolonged again
Stretch 5min, 4 DEG C of preservations.
It is attached:The sequence of Mse I suppressor primer:5'-GAG CAA GGC TCT CAC A-'3
The sequence of Pst I suppressor primer:5'-CTC GGA AGC CTC AGT C-'3
(5) the pre- amplification and detection of DNA sample
Pre- amplification reaction system：Takara Ex Taq 1U；10×Ex Taq Buffer 2μl；It has diluted
1 μ l of suppression PCR products；25mMMgC12 1.5μl；1.5 μ l of dNTP Mix (each 2.5mM)；Mse I adapter
Primer 1 5pmol； Pst I adapter primer 5pmol；DdH20 to 20 μ l.It is expanded by the following conditions.
It is attached：The sequence of Mse I adapter primer 1：5'-GAC GAC CGA CGA GTA AC-'3
The sequence of Pst I adapter primer：5'-AGA CTG CGT ACA TGC AGGA-'3
(6) SAM is expanded
SAM amplification reaction systems：10×Ex Taq Buffer 2μl；The 2 μ l of pre- amplified production diluted；Takara Ex
Taq lU；25mM MgCl2 1.5μl；1.5 μ l of dNTP Mix (each 2.5mM)；Mse I adapter primer 3 (4~10)
5pmol； SSR primer(PCT6)20pmol；Add ddH20 to 20 μ l, then presses table 1 and PCR amplification parameter is set.Amplified production
4 DEG C of storages are spare.
Table 1SAM PCR parameters
It is attached：The SSR primers of use --- the mixture that PCT6 is made of the oligonucleotide of two equimolar numbers.
Sequence is:5’-KKV RVR VCT CTC TCT CTC T’-3
5’-KKR VRV RTC TCT CTC TCT C’-3
The sequence of Mse I adapter primer 3：5'-GAC GAC CGA CGA GTA ACAA -‘3
The sequence of Mse I adapter primer 4：5'-GAC GAC CGA CGA GTA ACA C-‘3
The sequence of Mse I adapter primer 5：:5'-GAC GAC CGA CGA GTA ACA G-‘3
The sequence of Afse I adapter primer 6：5'-GAC GAC CGA CGA GTA ACA T-‘3
The sequence of Afse I adapter primer 7：5'-GAC GAC CGA CGA GTA ACT A-‘3
The sequence of Mse I adapter primer 8：5'-GAC GAC CGA CGA GTA ACT C-‘3
The sequence of Mse I adapter primer 9：5'-GAC GAC CGA CGA GTA ACT G-‘3
The sequence of Mse I adapter primer 10：5'-GAC GAC CGA CGAGTA ACT T-‘3
(7) recycling of SAM amplified productions
The band of clearly SAM amplified productions is cut, the centrifuge tube of 0.5ml is turned；0.2ml deionizations ddH is added2O soaks
Adhesive tape is steeped, after impregnating l min, with liquid-transfering gun suck dry moisture；The sterile ddH of 50 μ l is added after adhesive tape is smashed to pieces as possible2O impregnates
1h；3000rpm centrifuges 3min, and 2 μ l of supernatant is taken to carry out second of SAM amplification, reaction system and Amplification as template
With for the first time；Recycle secondary SAM amplified productions.
(8) clone of SAM amplified productions
Connection reaction
According to the specification of the kit of precious bioengineering (Dalian) Co., Ltd production, by program by PMD 18-T
Vector is connected with PCR recovery products.
The preparation of competent cell
The single bacterium colony newly cultivated with sterile toothpick picking from LB tablets is inoculated in 25ml LB liquid mediums, is being shaken
Swing 37 DEG C of constant temperature in incubator, the uninterrupted shaken cultivation 13h of 200rpm；It is transferred to culture solution, ice bath in the 10ml centrifuge tubes of sterilizing
Cooling 10min；4 DEG C, 8000rpm centrifuges 5min；Go supernatant, sediment that the CaC1 of the 100mM of 10ml precoolings is added2Solution；
Ice bath 30min, 4 DEG C, 8000rpm centrifuges 5min, removes supernatant；With the CaC1 of the 100mM of 600 μ l precoolings2Sediment is resuspended in solution,
4 DEG C save backup.
Conversion
10 μ l connection reaction solutions are added in containing 100 μ l E.coli XL1 competent cell 1.5ml centrifuge tubes, are mixed,
It is embedded to trash ice, ice bath 30min；42 DEG C of water baths are immediately placed in, Heat thermostability 90S is embedded to 1-2min in ice；Draw 400 μ l LB
Culture medium turns centrifuge tube, mixing, 37 DEG C of constant incubator, 200rpm shake cultures 1h；200 μ l conversion fluids are drawn under aseptic condition
The even spread on LB solid plates (containing 100 μ g/ml Amp), 37 DEG C of culture 12-16h of constant incubator after drying.Recon
Bacterium solution PCR identification
The centrifuge tube of 1ml LB liquid culture mediums (containing 100 μ g/ml Amp), is added single bacterium colony on conversion tablet, constant temperature incubation
37 DEG C of culture 12-16h of case；Reaction system:2 μ l of pre- amplified production, 10 × Ex Taq Buffer, 2 μ l, the dNTP diluted
Mix (each 2.5mM) 1.5 μ l, Takara Ex Taq lU, 1.5 μ l of 25mM MgCl2, Mse I adapter primer 3 (4
~10) 5pmol, SSR primer (PCT6) 20pmol, sterile ddH2020 μ l；4500rpm centrifuges 10sec after mixing, is put into
PCR amplification instrument is expanded by table 2PCR parameters, the agarose gel electrophoresis detection of product 1% after amplification.
The puncture culture of recon
2ml puncture tubes are such as LB solid mediums (containing 100 μ g/ml Amp), and after to be solidified, the bacterium solution of picking recon is inserted
Enter in culture medium for several times, 37 DEG C of incubators, overnight.Sequencing is completed by precious bioengineering (Dalian) Co., Ltd.
(9) screening of useful primer
According to sequencing result, special primer uses Primer Premier5.0 Software for Design.By Shanghai biotechnology
Services Co., Ltd synthesizes.
The genomic DNA for rolling up marchantia using fist, using special primer, establishes PCR reaction systems as template：Taq DNA
Genomic DNA (100ng/ μ l) 2 μ l, 10 × PCR Buffer (Sangon), 2 μ l, the 25mM of Polymerase 1U, fist volume marchantia
MgCI21.2 μ l, dNTP Mixture (each 10mM) 0.4 μ l, Special primer 5pmol, SSR primer (PCT6)
5pmol, sterile ddH2O20μl；5000rpm centrifuges 10S after mixing；PCR amplification, parameter such as table 2,2% agarose of amplified production
Electrophoresis detection.
Table 2PCR Amplifications
2. result
(1) extraction of the genomic DNA of fist volume marchantia
Ultraviolet specrophotometer measures the OD of the genomic DNA of the fist volume marchantia of the CTAB methods extraction of display improvement260/
OD280Ratio is in 1.80 or so, OD260/OD230Ratio is more than 2.0, shows that purity is relatively high, while through 1% Ago-Gel electricity
Swimming detects, and the genomic DNA of the fist volume marchantia after extraction purification is the band of a complete display, not visible without traction
RNA, and brightness is high.
(2) digestion of DNA and the detection of inhibition PCR, pre- amplified production
The genomic DNA for rolling up marchantia to fist with restriction enzyme Mse Ι and Pst Ι carries out double digestion, and the 1% of digestion products
Agarose gel electrophoresis detects, and Dispersed precipitate, clip size are concentrated mainly on about the DNA of double digestion in the form of sheets in swimming lane
Within the scope of 100-1500bp.Inhibition PCR product is rendered as high-visible disperse segment, size about 100-1000bp.Pre-expansion
The amplified production clip size of volume increase object concentrates on 150-800 or so.
(3) SAM amplifications are analyzed
SAM amplified productions are detached by native polyacrylamide gel electrophoresis, gel silver staining result such as Fig. 1:Almost
There is 10-30 items clearly band in each swimming lane of Polyacrylamide gradient gel, size is between 50-700bp, different swimming lanes
Amplified band due to be with Mse I adapter-primer of the 3 ' ends containing different selective bases amplify come, the DNA between each swimming lane
Bands of a spectrum are all different.
(4) recycling of SAM amplified productions and clone
Random to select 200 SAM amplified fragments of different sizes, pipette tips crushing, which is simultaneously impregnated with distilled water, to be centrifuged, and carries out the
Secondary SAM amplifications, amplified production are purified with agarose gel electrophoresis, the recycling of DNA purification kits, 2% fine jade of the product of recycling
Sepharose electrophoresis detection, each swimming lane only reveal a clearly band, clip size about 150bp.
Recovery product is transferred to competent cell by the connection with PMD 18-T Vector, is obtained through Amp+LB plate screenings
To positive colony, 2 white colonies are selected from each tablet at random.It is detected through bacterium solution PCR, the weight containing purposeful Insert Fragment
Group sends sequencing analysis after carrying out puncture culture.Clone has obtained 100 SAM segments for being sequenced in total.
(5) design of special primer
The design of fist volume marchantia SSR special primers is according to the flanking sequence that SSR contained in obtained DNA fragmentation is sequenced
Using 5.0 Software for Design special primers of Primer premier, some segments are containing dimer, hairpin structure between primer or because of side
Wing sequence is too short and can not design suitable primer, according to the one or both sides sequence design in the sites SSR from 86 segments
Special primer is synthesized.
(6) screening of useful primer
Using designed different SSR special primers, draw with 5 ' anchoring degenerate primer SSR primer (PCT6) compositions
Object pair, the genomic DNA for rolling up marchantia using fist detect through 2% agarose gel electrophoresis as template, filter out 30 pairs of clear bands
Special primer.
Claims (8)
1. a kind of method of screening fist volume marchantia SSR primers, it is characterised in that：This method extracts the genome of fist volume marchantia first
DNA, then carries out digestion and connects, expanded in advance again and the amplification of SAM methods, PCR screen punch volume marchantia SSR primers.
2. a kind of method of screening fist volume marchantia SSR primers according to claim 1, it is characterised in that：The extraction
The genomic DNA of fist volume marchantia is that the genome DNA sample for extracting fist in marchantia blade and rolling up marchantia is rolled up from fist using CTAB methods.
3. a kind of method of screening fist volume marchantia SSR primers according to claim 2, it is characterised in that：The digestion,
The genome DNA sample for being connected as rolling up fist with restriction enzyme Mse I and Pst I marchantia carries out double digestion and connection, obtains
It is cut and connection product to fist volume marchantia DNA enzymatic, fist volume marchantia DNA enzymatic is cut to be detected with 1% agarose gel electrophoresis of connection product,
Its clip size is 100-1500bp.
4. a kind of method of screening fist volume marchantia SSR primers according to claim 3, it is characterised in that：The pre-expansion
It increases to cut fist volume marchantia DNA enzymatic and be expanded in advance with connection product, obtain pre- amplified production, the pre- amplification condition is：
Takara Ex Taq 1U；10×Ex Taq Buffer 2μl；The 1 μ l of suppression PCR products diluted；
25mMMgC12 1.5μl；1.5 μ l of dNTP Mix (each 2.5mM)；Mse I adapter Primer 1 5pmol；Pst I
adapter primer 5pmol；DdH20 to 20 μ l；Wherein：
The sequence of Mse I adapter primer 1：5'-GAC GAC CGA CGA GTA AC-'3
The sequence of Pst I adapter primer：5'-AGA CTG CGT ACA TGC AGG A-'3.
5. a kind of method of screening fist volume marchantia SSR primers according to claim 4, it is characterised in that：The SAM methods
Amplification is to be in reaction system by pre- amplified production：10×Ex Taq Buffer 2μl；The 2 μ l of pre- amplified production diluted；
Takara Ex Taq lU；25mM MgCl2 1.5μl；1.5 μ l of dNTP Mix (each 2.5mM)；Mse I adapter primer
3 (4~10) 5pmol；SSR primer(PCT6)20pmol；Add ddH20 to 20 μ l, under conditions of carry out SAM amplifications, obtain
SAM amplified productions.
6. a kind of method of screening fist volume marchantia SSR primers according to claim 5, it is characterised in that：The SAM methods
Amplification further includes recycling and the clone of SAM amplified productions, and the recycling of the SAM amplified productions is with clone：By non denatured
Polyacrylamide gel electrophoresis detaches SAM amplified productions, selects 200 SAM amplified fragments of different sizes, pipette tips pressure at random
It is broken and impregnated and centrifuge with distilled water, second of SAM amplification is carried out, amplified production purified with agarose gel electrophoresis, DNA purifying examinations
Agent box recycles, and recovery product is transferred to competent cell by the connection with PMD18-TVector, is obtained through Amp+LB plate screenings
To positive colony, 2 white colonies are selected from each tablet at random, clone has obtained 100 SAM segments for being sequenced altogether.
7. a kind of method of screening fist volume marchantia SSR primers according to claim 6, it is characterised in that：The PCR sieves
It is selected as：The flanking sequence of SSR contained in the DNA fragmentation obtained according to sequencing is special using Primerpremier5.0 Software for Design
Different primer is synthesized from 86 segments according to the one or both sides sequence design special primer in the sites SSR, then with 5 ' anchors
Degenerate primer SSRprimer (PCT6) composition primer pairs are determined, using the genomic DNA of fist volume marchantia as template, through 2% agarose
Detected through gel electrophoresis filters out the special primer of 30 pairs of clear bands, and as fist rolls up marchantia SSR primers.
8. a kind of method of screening fist volume marchantia SSR primers according to claim 2, it is characterised in that：The PCR sieves
It is selected as：Further include measuring display with ultraviolet specrophotometer to be rolled up with fist after the genome DNA sample for getting fist volume marchantia
The OD260/OD280 ratios of the genomic DNA of marchantia, while detecting DNA purity through 1% agarose gel electrophoresis.
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| CA2447261A1 (en) * | 2001-05-16 | 2002-11-21 | Austrian Research Centers Gmbh-Arc | Method for analysing dna of sweetpotato |
| CN103820546A (en) * | 2014-02-19 | 2014-05-28 | 河南科技大学 | Peony genome DNA molecule marking method |
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| CA2447261A1 (en) * | 2001-05-16 | 2002-11-21 | Austrian Research Centers Gmbh-Arc | Method for analysing dna of sweetpotato |
| CN103820546A (en) * | 2014-02-19 | 2014-05-28 | 河南科技大学 | Peony genome DNA molecule marking method |
| CN105368930A (en) * | 2015-10-13 | 2016-03-02 | 中国农业大学 | Determining method for sequencing enzyme digestion combination in sequencing genotyping technology |
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