CN109652515A - A method of identifying Chinese wax new varieties gold arrow using Capillary Electrophoresis fluorescence SSR finger-print - Google Patents
A method of identifying Chinese wax new varieties gold arrow using Capillary Electrophoresis fluorescence SSR finger-print Download PDFInfo
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Abstract
The invention discloses a kind of methods for identifying Chinese wax new varieties ' golden arrow ' using Capillary Electrophoresis fluorescence SSR finger-print, it is that Chinese wax genomic DNA is extracted by Chinese wax young leaflet tablet, and using the DNA of extraction as template, PCR amplification is carried out using the SSR primer pair filtered out, pcr amplification product is subjected to Capillary Electrophoresis, according to the peak value of SSR primer each pair of in electrophoresis result, combination forms the SSR finger-print of Chinese wax new varieties ' golden arrow ', realizes the identification to Chinese wax new varieties ' golden arrow '.Using 6 pairs of good SSR special primers of specific stabilization, polymorphism are sifted out in the method for the present invention, construct the finger-print of Chinese wax new varieties ' golden arrow ', can it is easy, be completed rapidly and accurately the ' identification of golden arrow ' sample of Chinese wax new varieties.
Description
Technical field
The invention belongs to plant variety molecular identification technical fields, in particular to a kind of glimmering using Capillary Electrophoresis
The method that light SSR finger-print identifies Chinese wax new varieties ' golden arrow '.
Background technique
Chinese wax platymiscium is anemophilous flower, and when companion planting is easy to that open-pollinated hybridization, especially Fraxinus velutina occurs
(F.velutina), red Xin (F.pennsylvanica), white Xin (F.americana) etc. are all Xin subgenus sample group, and form is special
Levy closely similar, affiliation is extremely got close to, and the traditional method of filial generation often cannot be distinguished.In the past all when distinguishing seedling
It is but to be difficult to differentiate between that form is very much like or affiliation pole using macroscopical genetic analysis biological characteristics and morphological feature index
Close seedling, the breeding for especially planting interior different level can not even more identify, so that occur miscegenation in production, mixed system, mix strain, with
The case where vacation is looked genuine, result bring extreme loss to production.Germplasm is identified not by environment on a molecular scale and
The influence of growth phase, it is as a result stable, it is highly reliable.Therefore, seek really to accurately identify Chinese wax breeding from molecular level great
Realistic meaning.
There is simple repeated sequence (Simple5equencerepeat, SSR) molecular labeling information content height, codominance to lose
The advantages that biography, quantity are abundant, analysis method is simple, result is reproducible, time saving and energy saving economical, SSR marker is germplasm mirror at present
A kind of fixed most sensitive, the most reliable molecular labeling of aspect.And Capillary Electrophoresis fluorescence SSR molecular marker technology is utilized, develop base
Similar in the certain morphological features of Capillary Electrophoresis fluorescence SSR finger-print identification, affiliation gets close to the method for plant variety
Though having applied to the report in the crops such as corn, rice, soybean, wheat, cotton, it is applied to the correlation of forest especially Chinese wax
It reports actually rare.
Chinese wax new varieties ' golden arrow ' are Fraxinus velutina (FraxinusVelutina ' jinjian ') staminiferous plant, are by Shandong Province
What seminar, forestry scientific research institute cultivated, pass through country's authorization within 2016.The kind form is more straight, and saline-alkali tolerant is perennial, branch
Dry yellow, annual shoot yellow green, blade leathery and glossy, wide adaptation range.But ' golden arrow ' in seedling stage is difficult to pass through
Phenotypic characteristic is identified, made troubles to operator or is caused damages to production.Therefore, for Rapid identification Chinese wax new product
It plants ' golden arrow ', a kind of method for developing utilization Capillary Electrophoresis fluorescence SSR finger-print identification Chinese wax new varieties ' golden arrow ' has
Significance.
Summary of the invention
In view of the deficiencies of the prior art, the problem to be solved in the present invention is to utilize Capillary Electrophoresis fluorescence SSR for a kind of
The method that finger-print identifies Chinese wax new varieties ' golden arrow '.
The method of the present invention for identifying Chinese wax new varieties ' golden arrow ' using Capillary Electrophoresis fluorescence SSR finger-print, step
Suddenly it is:
(1) Chinese wax genomic DNA is extracted by Chinese wax young leaflet tablet；
(2) using the DNA of extraction as template, PCR amplification is carried out using the SSR primer pair filtered out；
(3) pcr amplification product is subjected to Capillary Electrophoresis；
(4) according to the peak value of SSR primer each pair of in electrophoresis result, combination forms the SSR fingerprint of Chinese wax new varieties ' golden arrow '
Map realizes the identification to Chinese wax new varieties ' golden arrow '；
It is characterized in that:
The extraction of DNA uses CTAB method in step (1), and 2% beta -mercaptoethanol and 2%PVP is added in Extraction buffer；It is purple
The concentration and purity of outer spectrophotometry measurement DNA, is diluted to 30ng μ l-1, -20 DEG C save, spare；
The SSR primer pair that step (2) filters out is 6 pairs, is F186 and R186 respectively, F187 and R187, F202 and
R202, F203 and R203, F208 and R208, F213 and 213 are wherein held in each pair of primer 5 ' added with selected from FAM, TAMRA, HEX
With any one fluorescent marker in ROX, forward primer is to form with the TP-M13 primer for carrying fluorescent marker；The F186 and
The nucleotide sequence of F186 is the forward primer sequence as shown in SEQ ID No.1, the nucleotides sequence of R186 in R186 primer pair
Column are the forward primer sequences as shown in SEQ ID No.2, and the nucleotide sequence of F187 is in F187 the and R187 primer pair
The forward primer sequence as shown in SEQ ID No.3, the nucleotide sequence of R187 are the forward primers as shown in SEQ ID No.4
Sequence, the nucleotide sequence of F202 is the forward primer sequence as shown in SEQ ID No.5 in F202 the and R202 primer pair,
The nucleotide sequence of R202 is the forward primer sequence as shown in SEQ ID No.6, F203 in F203 the and R203 primer pair
Nucleotide sequence be the forward primer sequence as shown in SEQ ID No.7, the nucleotide sequence of R203 is such as SEQ ID No.8
Shown in forward primer sequence, the nucleotide sequence of F202 is as shown in SEQ ID No.9 in F208 the and R208 primer pair
Forward primer sequence, the nucleotide sequence of R208 is the forward primer sequence as shown in SEQ ID No.10, the F213 and
The nucleotide sequence of F213 is the forward primer sequence as shown in SEQ ID No.11, the nucleotides sequence of R213 in R213 primer pair
Column are the forward primer sequences as shown in SEQ ID No.12；
PCR reaction system is 10 μ L:10ng. μ L-11 μ L of DNA profiling, 5 μ of 2X Taq plus PCR Master Mix
L、 10μmol·L-1Each 0.1 μ L and ddH of forward and reverse primer2O totally 10 μ L；
PCR amplification program uses Touchdown mode: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 56 DEG C of annealing 30s；
Each circulation reduces by 0.5 DEG C；72 DEG C of extension 30s, 15 circulations；95 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 30s, 20
A circulation；72 DEG C of extension 10min, 4 DEG C of preservations；
Step (3) method that pcr amplification product is carried out Capillary Electrophoresis is: taking 0.3 μ L of PCR product, GS-
PCR plate, 95 DEG C of denaturation 5min, after 4 DEG C of coolings are added in 0.5 μ L of 500LIZ molecular weight internal standard and 9.5 μ L of deionized formamide mixing
It is centrifuged, machine testing on 1 × Buffer buffer；Capillary Electrophoresis is carried out using 3730xl DNA sequencer: under prerunning 15kV
3min；Sample introduction 15s under 1.6kV；20min under electrophoresis 15kV；
Discrimination method described in step (4) is: carrying out data preparation and image point using GenemarkerZ.2.0 software
Analysis, according to the peak value of each pair of SSR primer, combination forms the SSR finger-print of Chinese wax new varieties ' golden arrow '；Wherein, F186 and
The peak value of R186 primer pair is 93/99, F187 and the peak value of R187 primer pair is the peak of 134/140, F202 and R202 primer pair
Value is 109/112, F203 and the peak value of R203 primer pair is 157, F208 and the peak value of R208 primer pair is 139/145, F213
Peak value with R213 primer pair is 81/90/96.
The invention also discloses the SSR primer pairs for the above method, it is characterized in that: the SSR primer pair is 6 pairs, point
It is not F186 and R186, F187 and R187, F202 and R202, F203 and R203, F208 and R208, F213 and 213 is wherein every
To the end of primer 5 ' added with FAM fluorescent marker, forward primer is to form with the TP-M13 primer for carrying fluorescent marker；The F186 and
The nucleotide sequence of F186 is the forward primer sequence as shown in SEQ ID No.1, the nucleotides sequence of R186 in R186 primer pair
Column are the forward primer sequences as shown in SEQ ID No.2, and the nucleotide sequence of F187 is in F187 the and R187 primer pair
The forward primer sequence as shown in SEQ ID No.3, the nucleotide sequence of R187 are the forward primers as shown in SEQ ID No.4
Sequence, the nucleotide sequence of F202 is the forward primer sequence as shown in SEQ ID No.5 in F202 the and R202 primer pair,
The nucleotide sequence of R202 is the forward primer sequence as shown in SEQ ID No.6, F203 in F203 the and R203 primer pair
Nucleotide sequence be the forward primer sequence as shown in SEQ ID No.7, the nucleotide sequence of R203 is such as SEQ ID No.8
Shown in forward primer sequence, the nucleotide sequence of F202 is as shown in SEQ ID No.9 in F208 the and R208 primer pair
Forward primer sequence, the nucleotide sequence of R208 is the forward primer sequence as shown in SEQ ID No.10, the F213 and
The nucleotide sequence of F213 is the forward primer sequence as shown in SEQ ID No.11, the nucleotides sequence of R213 in R213 primer pair
Column are the forward primer sequences as shown in SEQ ID No.12.Specifically, 6 couples of SSR primer pair information is shown in Table 1.
Table 1:6 is to SSR primer pair information
Application of the SSR primer pair of the present invention in the SSR finger-print of building Chinese wax new varieties ' golden arrow '.
The present invention designs and passes through according to the sequence data that transcript profile sequencing obtains and largely screens, and selects specific steady
6 pairs of SSR special primers fixed, polymorphism is good, for constructing the SSR finger-print of Chinese wax new varieties ' golden arrow '；With resolution ratio
The features such as high, easy to operate, quick, result is accurate, can fast implement ' the identification of golden arrow ' sample of Chinese wax new varieties.
The present invention provides a kind of SSR finger-prints of Chinese wax new varieties ' golden arrow ', it is characterized in that: the map is by above-mentioned
Identifying Chinese wax new varieties using Capillary Electrophoresis fluorescence SSR finger-print, ' correlation step in golden arrow ' method, which constructs, to be obtained.
In the SSR finger-print of above-mentioned Chinese wax new varieties ' golden arrow ', the peak value of SSR primers F 186 and R186 primer pair is
The peak value of 93/99, F187 and R187 primer pair be 134/140, F202 and the peak value of R202 primer pair be 109/112, F203 and
The peak value of R203 primer pair is 157, F208 and the peak value of R208 primer pair is the peak value of 139/145, F213 and R213 primer pair
It is 81/90/96.
Compared with prior art, excellent beneficial effect of the invention is embodied in:
1) method provided by the invention is that one kind is quick from molecular level, efficiently identifies Chinese wax new varieties method, is had
Quickly, accurately, the advantages such as easy to operate, and qualification result be not easy it is affected by environment.
2) present invention utilizes Capillary Electrophoresis fluorescent marker electrophoretic techniques, overcomes polyacrylamine gel electrophoresis not
Foot, can accurately read the size of product segment, and accuracy reaches within l bp, and accurate identification, and cost can be carried out to kind
Lower, application value is big.
3) the SSR finger-print of SSR primer pair and Chinese wax new varieties ' golden arrow ' provided by the invention is DNA fingerprinting
The further application study on Chinese wax provide solid theories integration.
Detailed description of the invention
Pcr amplification product Capillary Electrophoresis figure of Fig. 1: the SSR primer pair 186 in ' golden arrow '.
Pcr amplification product Capillary Electrophoresis figure of Fig. 2: the SSR primer pair 187 in ' golden arrow '.
Pcr amplification product Capillary Electrophoresis figure of Fig. 3: the SSR primer pair 202 in ' golden arrow '.
Pcr amplification product Capillary Electrophoresis figure of Fig. 4: the SSR primer pair 203 in ' golden arrow '.
Pcr amplification product Capillary Electrophoresis figure of Fig. 5: the SSR primer pair 208 in ' golden arrow '.
Pcr amplification product Capillary Electrophoresis figure of Fig. 6: the SSR primer pair 213 in ' golden arrow '.
Specific embodiment
Embodiment 1
1. expert evidence
Select ' golden arrow ' young leaflet tablet extraction genomic DNA.
The extraction of 2.DNA
Prepared CTAB Extraction buffer is placed in 65 DEG C of water-bath and preheats 30min by 2.1, one isoamyl alcohol of chloroform
(24:1), dehydrated alcohol, 70% ethyl alcohol are placed in the pre- cold standby of -20 DEG C of refrigerators, and mortar need to be pre-chilled in advance, using preceding again with a small amount of
Liquid nitrogen precooler；
2.2 take 0.4-0.5g Chinese wax spire, and sufficient liquid nitrogen grinding is added to be transferred to rapidly in 2mL centrifuge tube to powdered, are added
The CTAB extracting solution (containing 2% beta -mercaptoethanol) of 600 μ L 2%, mixes well.The water-bath 30min in 65 DEG C of water-bath,
Between gently overturn 3-4 times；
2.3 are cooled to the chloroform that 600 μ L are added after room temperature: isoamyl alcohol (24:1) extracts 10min, ceaselessly lightly shakes therebetween
It is dynamic, it is cooled to room temperature, is centrifuged 10min in 12000r/min；
2.4 are transferred to supernatant (about 500 μ l) in another centrifuge tube, and add isometric chloroform: isoamyl alcohol (24:l) repeats to walk
Rapid 2.3；
2.5 taking-up supernatants simultaneously moves on in the centrifuge tube of another clean 1.5ml, then plus 1ml pre-cooling dehydrated alcohol
It precipitates DNA (- 20 DEG C of placement 30min), gently rotating centrifugal pipe, cotton-shaped DNA, -20 DEG C of placement 1h occurs, observation precipitating generates；
2.6 8000rmp room temperature is centrifuged 5min, supernatant is outwelled, precipitating uses the ethanol wash of 1mL 75% twice, ultra-clean
It is clean that workbench stands ethyl alcohol volatilization.
2.7 after DNA is air-dried (the unsuitable overdrying of DNA, otherwise extremely difficult dissolution), with suitable TE (pH8.0,50 μ l)
Or ultrapure water dissolution, 4 DEG C of placement 6-12h dissolve it sufficiently；
2.8 are added 2 μ L RNA enzyme 10mg/mL, and 37 DEG C of water-bath 1h, -80 DEG C save backup；
3.DNA is quantified and quality testing
3.1 take 2 μ L DNA to be detected with trace dna Protein Detection instrument, can directly read DNA concentration and A260/A280
Ratio, ratio meet the requirements between 1.8~2.0.
3.2 bromophenol blues for separately 3 μ L DNA being taken to add 1 μ L, electrophoresis 15min~20min, pressure stabilizing on 0.8% Ago-Gel
100V, electrophoretic buffer are 1 × TBE, observe under 325nm ultraviolet lamp, take a picture, be compared and estimated by the fluorescence with standard solution
Survey the concentration of unknown DNA.
3.3 plant genomic DNA samples are presented that a relative molecular weight is larger and the clear band of migration rate very little.
Sample is diluted to concentration as 20ng/ μ L by 3.4, is saved backup at -80 DEG C.
4. capillary electrophoresis system detects
4.1 screen one by one in well known 196 parts of resources, and selecting 6 pairs of good SSR primers of polymorphism, (details are shown in Table
1), addition fluorescent marker FAM (6-carboxy-fluorescein) is held in each pair of primer 5 '.Forward primer is and carries fluorescence
The TP-M13 primer of label forms, and is synthesized by bok gene Engineering Co., Ltd, Shandong China.
Table 1:6 is to SSR primer pair information
The PCR amplification of 4.2 fluorescent marker Capillary Electrophoresis uses the reaction system of 10 μ L: 10ng. μ L-11 μ of DNA profiling
L, 5 μ L of 2X Taq plus PCR Master Mix, each 0.1 μ L of forward and reverse primer (10 μm of olL-1) and ddH2O totally 10 μ
L。
4.3PCR amplification program uses Touchdown mode: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 56 DEG C of annealing
30s (each circulation reduces by 0.5 DEG C), 72 DEG C of extension 30s, 15 circulations；95 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C extend
30s, 20 circulations；72 DEG C of extension 10min, 4 DEG C of preservations.
The denaturation of 4.4PCR product: PCR product dilutes 10-20 times with distilled water, takes 0.3 μ L of PCR product, molecular weight internal standard
0.5 μ L and 9.5 μ L of deionized formamide, which is mixed, is added PCR plate；Run denaturation program in PCR instrument: 95 DEG C of denaturation 5min, 4 DEG C
It is centrifuged after cooling, machine testing on 1 × Buffer buffer.
4.5PCR product amplification detection: the 3730xl DNA that PCR product detection is produced using American AB I company
Analyzer Genetic Analyser, the 50cm produced using American AB I company, 96 Capillary Electrophoresis, 3min under prerunning 15kV；
Sample introduction 15s under 1.6kV；20min under electrophoresis 15kV.
Upper machine result original document is imported into Genemarker 2.2.0 software, carries out data collection and image analysis.
5. data are analyzed
The molecular size range and primer color that determine when according to screening primer carry out the determination of molecular weight, by the original of collection
Data import Gene Marker 2.2.0 system using Data Collection software and are analyzed.According to target peak size
System software is compared with the GS-500LIZ molecular weight internal standard in its swimming lane, accurately calculates target DNA fragments size, will
Electrophoresis result is converted to the export of PDF picture format.
3 repetitions of each fluorescent marker capillary electrophoresis detection, 3 duplicate average values are as experimental material on the seat
Data.
6.SSR fingerprint map construction and Molecular Identification:
According to the peak value size of 6 pairs of SSR primers, the SSR finger-print of Chinese wax new varieties ' golden arrow ' is constructed, is shown in Table 2.
The SSR finger-print of the Chinese wax new varieties ' golden arrow ' of 26 pairs of SSR fluorescent primer combination buildings of table
In the SSR finger-print of above-mentioned Chinese wax new varieties ' golden arrow ', the peak value of SSR primers F 186 and R186 primer pair is
The peak value of 93/99, F187 and R187 primer pair be 134/140, F202 and the peak value of R202 primer pair be 109/112, F203 and
The peak value of R203 primer pair is 157, F208 and the peak value of R208 primer pair is the peak value of 139/145, F213 and R213 primer pair
It is 81/90/96.
Sequence table
<110>Shandong Forest Science Academy Shandong China bok gene engineering science and technology Co., Ltd
<120>a kind of method for identifying Chinese wax new varieties gold arrow using Capillary Electrophoresis fluorescence SSR finger-print
<141> 2018-12-20
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence
<221>186 nucleotide sequence of primers F
<400> 1
tcttcacgtc ttctgtttgt tca 23
<210> 2
<211> 23
<212> DNA
<213>artificial sequence
<221>primer R186 nucleotide sequence
<400> 2
gaaaacgtgt gaatgagttt ggt 23
<210> 3
<211> 23
<212> DNA
<213>artificial sequence
<221>187 nucleotide sequence of primers F
<400> 3
tcgatctttc catctaaaca agc 23
<210> 4
<211> 23
<212> DNA
<213>artificial sequence
<221>primer R187 nucleotide sequence
<400> 4
aacgtgtgaa tgagtttggt ttt 23
<210> 5
<211> 23
<212> DNA
<213>artificial sequence
<221>202 nucleotide sequence of primers F
<400> 5
agttttcacc gctttcagtg tta 23
<210> 6
<211> 23
<212> DNA
<213>artificial sequence
<221>primer R202 nucleotide sequence
<400> 6
gggaatgaac atgagtttca gta 23
<210> 7
<211> 23
<212> DNA
<213>artificial sequence
<221>203 nucleotide sequence of primers F
<400> 7
gttatcagta gatgcaaccg cac 23
<210> 8
<211> 22
<212> DNA
<213>artificial sequence
<221>primer R203 nucleotide sequence
<400> 8
aacaccggtt ttcaacattt ct 22
<210> 9
<211> 23
<212> DNA
<213>artificial sequence
<221>208 nucleotide sequence of primers F
<400> 9
cctcctattg aatcattcgc tta 23
<210> 10
<211> 23
<212> DNA
<213>artificial sequence
<221>primer R208 nucleotide sequence
<400> 10
attttgattt ccctcctctg aag 23
<210> 11
<211> 23
<212> DNA
<213>artificial sequence
<221>213 nucleotide sequence of primers F
<400> 11
gacaacatgc ctaaattgga ctc 23
<210> 12
<211> 23
<212> DNA
<213>artificial sequence
<221>primer R213 nucleotide sequence
<400> 12
aattctgaac ttcaaggtgg gat 23
Claims (5)
1. a kind of method for identifying Chinese wax new varieties ' golden arrow ' using Capillary Electrophoresis fluorescence SSR finger-print, step is:
(1) Chinese wax genomic DNA is extracted by Chinese wax young leaflet tablet；
(2) using the DNA of extraction as template, PCR amplification is carried out using the SSR primer pair filtered out；
(3) pcr amplification product is subjected to Capillary Electrophoresis；
(4) according to the peak value of SSR primer each pair of in electrophoresis result, combination forms the SSR finger-print of Chinese wax new varieties ' golden arrow ',
Realize the identification to Chinese wax new varieties ' golden arrow '；
It is characterized in that:
The extraction of DNA uses CTAB method in step (1), and 2% beta -mercaptoethanol and 2%PVP is added in Extraction buffer；Ultraviolet point
Light photometry measures the concentration and purity of DNA, is diluted to 30ng μ l-1, -20 DEG C save, spare；
The SSR primer pair that step (2) filters out is 6 pairs, is F186 and R186 respectively, F187 and R187, F202 and R202, F203
And R203, F208 and R208, F213 and 213 are wherein held in each pair of primer 5 ' added with appointing in FAM, TAMRA, HEX and ROX
It anticipates a kind of fluorescent marker, forward primer is to form with the TP-M13 primer for carrying fluorescent marker；F186 the and R186 primer pair
The nucleotide sequence of middle F186 is the forward primer sequence as shown in SEQ ID No.1, and the nucleotide sequence of R186 is such as SEQ
Forward primer sequence shown in ID No.2, the nucleotide sequence of F187 is such as SEQ ID in F187 the and R187 primer pair
Forward primer sequence shown in No.3, the nucleotide sequence of R187 is the forward primer sequence as shown in SEQ ID No.4, described
The nucleotide sequence of F202 is the forward primer sequence as shown in SEQ ID No.5, the core of R202 in F202 and R202 primer pair
Nucleotide sequence is the forward primer sequence as shown in SEQ ID No.6, the nucleotide of F203 in F203 the and R203 primer pair
Sequence is the forward primer sequence as shown in SEQ ID No.7, the nucleotide sequence of R203 be as shown in SEQ ID No.8 just
To primer sequence, the nucleotide sequence of F202 is that the forward direction as shown in SEQ ID No.9 is drawn in F208 the and R208 primer pair
Object sequence, the nucleotide sequence of R208 are the forward primer sequence as shown in SEQ ID No.10, F213 the and R213 primer
The nucleotide sequence of centering F213 is the forward primer sequence as shown in SEQ ID No.11, the nucleotide sequence of R213 be as
Forward primer sequence shown in SEQ ID No.12；
PCR reaction system is 10 μ L:10ng. μ L-11 μ L of DNA profiling, 5 μ L of 2X Taq plus PCR Master Mix, 10 μ
mol·L-1Each 0.1 μ L and ddH of forward and reverse primer2O totally 10 μ L；
PCR amplification program uses Touchdown mode: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 56 DEG C of annealing 30s；Each
Circulation reduces by 0.5 DEG C；72 DEG C of extension 30s, 15 circulations；95 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 30s, 20 are followed
Ring；72 DEG C of extension 10min, 4 DEG C of preservations；
Step (3) method that pcr amplification product is carried out Capillary Electrophoresis is: taking 0.3 μ L of PCR product, GS-500LIZ points
PCR plate is added in son amount 0.5 μ L of internal standard and 9.5 μ L of deionized formamide mixing, and 95 DEG C of denaturation 5min are centrifuged after 4 DEG C of coolings, 1 ×
Machine testing on Buffer buffer；Capillary Electrophoresis: 3min under prerunning 15kV is carried out using 3730xl DNA sequencer；
Sample introduction 15s under 1.6kV；20min under electrophoresis 15kV；
Discrimination method described in step (4) is: carrying out data preparation and image analysis, root using GenemarkerZ.2.0 software
According to the peak value of each pair of SSR primer, combination forms the SSR finger-print of Chinese wax new varieties ' golden arrow '；Wherein, F186 and R186 primer
Pair peak value be 93/99, F187 and the peak value of R187 primer pair is 134/140, F202 and the peak value of R202 primer pair is 109/
The peak value of 112, F203 and R203 primer pair is 157, F208 and the peak value of R208 primer pair is that 139/145, F213 and R213 draws
The peak value of object pair is 81/90/96.
2. it is used for the SSR primer pair of claim 1 the method, it is characterized in that: the SSR primer pair is 6 pairs, it is F186 respectively
And R186, F187 and R187, F202 and R202, F203 and R203, F208 and R208, F213 and 213 are wherein in each pair of primer 5 '
Added with FAM fluorescent marker, forward primer is to form with the TP-M13 primer for carrying fluorescent marker at end；F186 the and R186 primer
The nucleotide sequence of centering F186 is the forward primer sequence as shown in SEQ ID No.1, and the nucleotide sequence of R186 is such as SEQ
Forward primer sequence shown in ID No.2, the nucleotide sequence of F187 is such as SEQ ID in F187 the and R187 primer pair
Forward primer sequence shown in No.3, the nucleotide sequence of R187 is the forward primer sequence as shown in SEQ ID No.4, described
The nucleotide sequence of F202 is the forward primer sequence as shown in SEQ ID No.5, the core of R202 in F202 and R202 primer pair
Nucleotide sequence is the forward primer sequence as shown in SEQ ID No.6, the nucleotide of F203 in F203 the and R203 primer pair
Sequence is the forward primer sequence as shown in SEQ ID No.7, the nucleotide sequence of R203 be as shown in SEQ ID No.8 just
To primer sequence, the nucleotide sequence of F202 is that the forward direction as shown in SEQ ID No.9 is drawn in F208 the and R208 primer pair
Object sequence, the nucleotide sequence of R208 are the forward primer sequence as shown in SEQ ID No.10, F213 the and R213 primer
The nucleotide sequence of centering F213 is the forward primer sequence as shown in SEQ ID No.11, the nucleotide sequence of R213 be as
Forward primer sequence shown in SEQ ID No.12.
3. application of the SSR primer pair as claimed in claim 2 in the SSR finger-print of building Chinese wax new varieties ' golden arrow '.
4. a kind of SSR finger-print of Chinese wax new varieties ' golden arrow ', it is characterized in that: the map is by claim 1 the method
Building obtains.
5. the SSR finger-print of Chinese wax new varieties ' golden arrow ' according to claim 4, it is characterized in that: the SSR fingerprint image
In spectrum, the peak value of SSR primers F 186 and R186 primer pair is 93/99, F187 and the peak value of R187 primer pair is 134/140,
The peak value of F202 and R202 primer pair is 109/112, F203 and the peak value of R203 primer pair is 157, F208 and R208 primer pair
Peak value be 139/145, F213 and the peak value of R213 primer pair is 81/90/96.
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| CN104164503A (en) * | 2014-08-04 | 2014-11-26 | 山东省林业科学研究院 | Method for identifying fraxinus chinensis species by virtue of SSR (Simple Sequence Repeat) fingerprint spectrum |
| CN105861643A (en) * | 2015-12-03 | 2016-08-17 | 山东农业工程学院 | Construction method for fraxinus velutina SSR marked molecular genetic linkage map |
| CN106244700A (en) * | 2016-08-24 | 2016-12-21 | 山东省林业科学研究院 | A kind of method utilizing SSR marker technology that Fraxinus velutina and American Red cenospecies are carried out Rapid identification |
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| CN104164503A (en) * | 2014-08-04 | 2014-11-26 | 山东省林业科学研究院 | Method for identifying fraxinus chinensis species by virtue of SSR (Simple Sequence Repeat) fingerprint spectrum |
| CN105861643A (en) * | 2015-12-03 | 2016-08-17 | 山东农业工程学院 | Construction method for fraxinus velutina SSR marked molecular genetic linkage map |
| CN106244700A (en) * | 2016-08-24 | 2016-12-21 | 山东省林业科学研究院 | A kind of method utilizing SSR marker technology that Fraxinus velutina and American Red cenospecies are carried out Rapid identification |
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