CN106521015A - SRAP marking method with fluorescent label - Google Patents
SRAP marking method with fluorescent label Download PDFInfo
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- CN106521015A CN106521015A CN201611256864.5A CN201611256864A CN106521015A CN 106521015 A CN106521015 A CN 106521015A CN 201611256864 A CN201611256864 A CN 201611256864A CN 106521015 A CN106521015 A CN 106521015A
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Abstract
The invention discloses an SRAP marking method with a fluorescent label. A forward primer is a primer with the fluorescent label. An FSRAP detection mode is good in stability, rich in polymorphism among varieties, high in resolution ratio and good in application prospect.
Description
Technical field
The present invention relates to a kind of marks of the SRAP with fluorescence labels (Fluorescent SRAP, FSRAP) method.
Background technology
SRAP (Sequence-related amplified polymorphism) is calendar year 2001 California, USA university
Li and Quiros [Li et al.Theo Appl Genet, 2001,103:455-461] molecule of a kind of PCR-based developed
Mark, it overcomes the shortcoming of the marks such as RFLP, RAPD, SSR and AFLP, such as:RFLP is marked to DNA concentration, the requirement of purity
It is high；There is migration altogether in RAPD, it is impossible to differentiate heterozygote and homozygote, stability and poor repeatability, and low yield；The cost of AFLP
Expensive and step is complicated；The primer development of SSR is the shortcomings of time-consuming.Therefore, SRAP molecular labelings have it is economical, easy to operate,
Yield is high, stable and is easy to the characteristics of cloning target segment, is widely used to analysis of genetic diversity, Germplasm Identification, compares
Structure of genomics and genetic linkage map etc. is studied.
SRAP molecular labelings are by specific primer to ORFs (Open reading frames, open reading frame)
Design is expanded.Research shows that gene extron G, C content enrich, and intron A, T enrich.The total forward direction of SRAP molecular labelings
With reverse two sets of primers." CCGG " sequence used in forward primer, is in order to make in specific bond ORFs region
Extron.Typically guarded due to Different Individual Exon sequence, low polymorphism level, this causes extron do
For mark.Region rich in AT is typically occurred in promoter and introne, and the core sequence of reverse primer is AATT, specific
Area is rich in reference to AT, the SRAP polymorphism marks based on introne and extron are amplified.Different species, different individualities,
Due to introne, promoter it is different from gap length, and produce polymorphism amplified production.
Primer is the key of SRAP molecular labelings.A pair of SRAP molecular labeling primers used in PCR reaction systems：Forward direction is drawn
The long 17bp of thing, the front 10bp at 5` ends is one section of nonspecific padding sequence, and followed by CCGG, they constitute core sequence together
Row, followed by be 3 selective bases against 3`, 3 optionally base can be change, its change can be produced
A series of primer, they have common core sequence.The long 18bp of reverse primer, i.e., by 11 fillings without specificity of 5`
The core sequence of sequence and back to back AATT compositions, and 3 selective bases of 3`, are carried out to introne and promoter region
Specific amplified.
SRAP molecular labelings are developed in rape crop earliest, up to the present, SRAP mark wheat,
It is used widely in the analysis of the Genetic Diversities such as paddy rice, arabidopsis, cotton, potato, tomato, capsicum, celery, has
The advantages of reproducible, easy to operate, codominance is high, band is easily separated, be adapted to genetic map construction, analysis of genetic diversity,
The biological studies such as the characterization and evaluation of germ plasm resource, important character genetic marker.
Inventor started with genetic diversity, the evaluation of NIL and the heredity that SRAP marks are carried out from 2008
Structure of collection of illustrative plates etc. is studied, successively affiliation [Ou Yangzhongming, Zhao of Chinese Pinellia using SRAP marker research
It is quiet, Peng Zhengsong, Yang Jun, Wei Shuhong. the SRAP analyses of Chinese Pinellia affiliation, plant science journal, 2012,30
(2):116-121.]；Wheat three gynoecium NIL [Yang Jun, Peng Zhengsong, Zhou Yonghong, Peng be have rated using SRAP marks
It is beautiful beautiful, Wei Shuhong. using SRAP molecular labelings evaluate three gynoecium NIL of wheat genetic background, 2012,26 (1):22-
27.]；And three gynobase of wheat is located because of Pis1 [article receives] using SRAP marks.Through years of researches, invention
People has found, although SRAP marks have the advantages that numerous, but operate still than relatively time-consuming, and this mainly receives silver staining and development
Time-consuming longer impact, while plain polypropylene acrylamide gel electrophoresis generally uses the glue of 1mm, resolution ratio and stability are all less
It is preferable.
Therefore, develop simple to operate, high resolution and good stability SRAP mark (Fluorescent SRAP,
FSRAP great meaning is respectively provided with to genetic diversity Journal of Sex Research, the structure of genetic map and molecular mark).
The content of the invention
In order to solve the above problems, the invention provides a kind of new mark (Fluorescent of the SRAP with fluorescence labels
SRAP, FSRAP) method.
SRAP labeling methods with fluorescence labels of the invention, its forward primer is the primer with fluorescence labels.
Preferably, the fluorescence labels are IRDye 700 or IRDye 800.
The step of labeling method, is as follows：
(1) extract the DNA in sample to be checked；
(2) with forward primer be the primer pair with fluorescence labels enter performing PCR amplification；
(3) result detection：DNA cloning result is detected.
In step (2), the system of the amplification is：1×PCR buffer,Mg2+1.8mmol/L, dNTP0.15mmol/L,
The each 0.4 μm of ol/L of forward and reverse primer, DNA profiling 20ng, Taq enzyme 1U, reaction system are 10 μ L.
In step (2), the program of the PCR amplifications is：94 DEG C of denaturations 5min；94 DEG C of denaturation 40s, 35 DEG C of renaturation 60s,
72 DEG C of extension 60s, totally 5 circulations；94 DEG C of 40s, 50 DEG C of 60s, 72 DEG C of 1min, totally 35 circulations；72 DEG C of continuation extension 10min, 4
DEG C insulation.
In step (3), the method for the detection is polyacrylamide gel electrophoresis, silver staining electrophoresis or utilizes genetic analysis system
The method of system LI-COR4300 electrophoresis.
Present invention also offers a kind of method of identification wheat breed MY29 and MY29TP, it is using using SEQ ID
(i.e. with primer shown in SEQ ID NO.3 as forward primer, primer shown in SEQ ID NO.6 is primer pair 1 shown in NO.3～6
Reverse primer) or SEQ ID NO.15～6 shown in primer pair 2 (i.e. with primer shown in SEQ ID NO.15 as forward primer,
Primer shown in SEQ ID NO.6 is reverse primer), detected according to preceding method.
FSRAP detection modes of the present invention rich polymorphism, high resolution between good stability, kind, while can be effective
The different cultivars of wheat is distinguished, detection is quick, and application prospect is good.
The present invention is described in further details below by specific embodiment, but is not the limit to the present invention
System, the above of the invention, according to the ordinary technical knowledge and customary means of this area, above-mentioned without departing from the present invention
Under the premise of basic fundamental thought, the modification of other various ways can also be made, is replaced or is changed.
Description of the drawings
Fig. 1 FSRAP forward primer structures
Fig. 2 FSRAP are marked and common SRAP marks compare.A:Common SRAP marks result, B:FSRAP marks result
Fig. 3 primers combine me1-em30 amplifications.M:Marker, arrow position represent differential band
Fig. 4 marks the wheat genetic collection of illustrative plates for building using FSRAP
Fig. 5 utilizes FSRAP Marker Identification wheat breed MY29 and MY29TP
Specific embodiment
1st, in the following example, experiment material, reagent and instrument used are as follows：
(1) experiment material
Wheat cultivar Mianyang 29 (MY29) by Sichuan Academy of Agricultural Sciences's biotechnology and Nuclear Techniques poplar
Wu Yun researcher provides.F2 colonies (totally 200 lists of wheat three gynoecium NIL MY29TP and MY29 × MY29TP hybridization
Strain), it is inventor place laboratory culture.
(2) agents useful for same
Plant DNA extraction kit (polysaccharide polyphenol sample) is Biotechnology brands；50-700bp DNA
Marker is purchased from into the rich bio tech ltd in Dulan；Primer is synthesized by Shanghai Ying Jun Bioisystech Co., Ltd；PCR reacts
Required reagent is purchased from precious biology (Dalian) Co., Ltd.
(3) instrument
BIO-RAD T100PCR instrument is BIO-RAD Products；2000 ultramicron spectrophotometrics of NanoDrop are
Thermo Products；LI-COR4300 genetic analysis systems are LI-COR Products.
2nd, DNA is extracted
29 (MY29) are extracted using plant DNA extraction kit (Biotechnology), MY29, MY29 × MY29TP are miscellaneous
The DNA of the F2 colonies (200 individual plants) of friendship, concrete operations are with reference to kit specification.With 2000 ultramicron light splitting of NanoDrop
The concentration and purity of photometer detection DNA.The OD A260/A280 values of DNA solution should be 1.8-2.0.
3rd, primer sequence
In the present embodiment, as shown in table 1, the 5` ends of forward primer add fluorescence labels IRDye to the primer sequence
700 or IRDye 800.
The primer sequence in 1 the present embodiment of table
1 FSRAP of embodiment marks the comparison with common SRAP marks
1st, experimental technique
To verify that FSRAP marks are marked better than common SRAP, with MY29 and MY29TP as experiment material, from same primers
The FSRAP primers and SRAP primers of sequence expands the genomic DNA of MY29 and MY29TP simultaneously, then compares both primers
Amplified band.
The experiment flow of common SRAP marks carries out [Yang Jun, Peng Zhengsong, Zhou Yong with reference to the article delivered before inventor
It is red, Peng Lijuan, Wei Shuhong. using SRAP molecular labelings evaluate three gynoecium NIL of wheat genetic background, 2012,26
(1):22-27.]。
The PCR system of FSRAP is completely the same with the PCR system of common SRAP, simply changes forward primer into band fluorescence mark
The primer of label.Specially：PCR system includes：1 × PCR buffer, Mg2+1.8mmol/L, dNTP 0.15mmol/L, it is positive and negative
To each 0.4 μm of ol/L of primer, DNA profiling 20ng, Taq enzyme 1U, reaction system are 10 μ L.PCR programs are：94 DEG C of denaturations
5min；94 DEG C of denaturation 40s, 35 DEG C of renaturation 60s, 72 DEG C of extension 60s, totally 5 circulations；94 DEG C of 40s, 50 DEG C of 60s, 72 DEG C of 1min,
Totally 35 circulations；72 DEG C are continued to extend 10min, 4 DEG C of insulations.
The detection of FSRAP marks is carried out using 4300 genetic analysis systems of LI-COR.It is prepared by PAGE glue；Dry in the air cleaning
Dry BOROFLOAT glass is assembled and is separated with edge strip (0.25mm), is carefully inserted in glue folder (GIBCO/BRL).Prepare
After ready, with syringe, by 25ml denaturant gel mixed liquors, (8% polyacrylamide adds 200 μ l10% ammonium persulfates before encapsulating
With 20 μ l TEMED, and rapidly mix) slowly inject from glue ogival sections be finally inserted comb and plus clip protection, cohesion 2h.
Electrophoretic separation；Offset plate is gripped out from glue, comb is taken out and is reversely inserted, clean glass outer side and be fixed on electrophoresis tank, on
Lower groove respectively adds 1 × tbe buffer liquids of 500ml, and the 1500V voltage prerunning 25min that switch on power are 30mA to electric current, are increased production in PCR
Isopyknic StopBuffer is added in thing, the 10min of ice bath cooling immediately after 94 DEG C of denaturation 3min.Prerunning finishes rear flush points
Sample hole, is loaded 0.5-0.7 μ l, 1500V constant pressure electrophoresis 3h or so per hole, passes through LI-COR4300 genetic analyses system in electrophoresis process
The infrared detector of system carries out real-time detection to amplified production, and electrophoresis terminates laggard line number according to statistics.
2nd, experimental result
With MY29 and MY29TP as experiment material, me2-em13 is combined using primer, me32-em13, me32-em14 are carried out
SRAP and FSRAP amplifications, amplified production are utilized respectively plain polypropylene acrylamide gel electrophoresis, silver staining and utilize genetic analysis systems
LI-COR4300 electrophoresis, each material are repeated 3 times.As a result as shown in Fig. 2 the band number of FSRAP amplifications is more than common SRAP,
And resolution ratio also more common SRAP mark is high, such as：Using FSRAP marks me2-em13, this has in MY29 and MY29TP to primer
Two difference sites, and common SRAP marks then do not see difference.
Experimental result illustrates that the sensitivity of FSRAP detection modes of the present invention is higher, the higher bar for more effectively expanding correlation
Band, high resolution.
The study on the stability of 2 FSRAP of embodiment marks
In order to verify FSRAP mark stability, with identical primer (primer combine me2-em13, me32-em13,
Me32-em14), the genomic DNA of MY29 and MY29TP is expanded, is repeated 3 times, compare amplified band with the presence or absence of difference, PCR is anti-
Should be with electrophoresis concrete operations with reference to embodiment 1.As shown in Figure 2 B, same material is carried out study on the stability result using same primers
3 PCR amplifications, the band for amplifying are completely the same, and the stability of this explanation FSRAP marks is preferable.
Embodiment 3 marks the genetic map for building wheat using FSRAP
The FSRAP primers that have differences in MY29 and MY29TP of screening, using the FSRAP marks for filtering out to MY29 ×
The F of MY29TP hybridization2Performing PCR amplification is entered by colony, and amplified production Jing after the detection of LI-COR4300 genetic analysis systems, go on business by statistics
Different band, PCR reactions and electrophoresis concrete operations are with reference to embodiment 1.Lost using the FSRAP of 4.0 software building wheats of JoinMap
Blit is composed.
Electrophoresis result is as shown in Figure 3.Having 13 FSRAP marks upper figure, this 13 marks to be belonging respectively to 4 linkage groups,
Wherein most is the 2nd linkage group, is marked containing 5；Minimum is the 4th linkage group, only 2 marks (Fig. 4).
Embodiment 4 utilizes FSRAP Marker Identification wheat breed MY29 and MY29TP
MY29 and MY29TP is the system such as near, and the two has similar economical character, therefore cannot pass through form before heading
Learn feature to identify MY29 and MY29TP, for this purpose, we develop the FSRAP suitable for MY29 and MY29TP seedling stage assays
Mark.Primer sequence for identifying FSRAP marks is as shown in table 2.
It is used for the FSRAP labeled primers that MY29 and MY29TP is identified in 2 the present embodiment of table
| Primer is combined | Forward primer Me | Reverse primer Em |
| me2-em13 | IRDye700-TGAGTCCAA ACCGGAGC | GACTGCGTACGAATTACG |
| me32-em13 | IRDye700-TGAGTCCAA ACCGGGCT | GACTGCGTACGAATTACG |
Electrophoresis result is as shown in figure 5, the combination of me2-em13 primers can amplify the specific bar of 100bp or so in MY29
Band, amplifies 150bp specific bands in MY29TP.The combination of me32-em13 primers can amplify 350bp or so in MY29
Specific band.Prove that these two pair primer amplified band in MY29 and MY29TP is highly stable through test is repeated several times,
Therefore can be used for the identification of MY29 and MY29TP.The present embodiment provide only a kind of wheat breed authentication method, using the method
The identification of Yield Traits of Wheat, disease resistance, resistance can also be carried out.
To sum up, FSRAP detection modes of the present invention rich polymorphism, high resolution between good stability, kind, while can be with
The different cultivars of wheat is distinguished effectively, detection is quick, and application prospect is good.
SEQUENCE LISTING
<110>China West Normal University
<120>A kind of SRAP labeling methods with fluorescence labels
<130> GY493-16P1614
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213> Me1
<400> 1
tgagtccaaa ccggata 17
<210> 2
<211> 18
<212> DNA
<213> Em5
<400> 2
gactgcgtac gaattaac 18
<210> 3
<211> 17
<212> DNA
<213> Me2
<400> 3
tgagtccaaa ccggagc 17
<210> 4
<211> 18
<212> DNA
<213> Em8
<400> 4
gactgcgtac gaattctg 18
<210> 5
<211> 17
<212> DNA
<213> Me4
<400> 5
tgagtccaaa ccggacc 17
<210> 6
<211> 18
<212> DNA
<213> Em13
<400> 6
gactgcgtac gaattacg 18
<210> 7
<211> 17
<212> DNA
<213> Me6
<400> 7
tgagtccaaa ccggtaa 17
<210> 8
<211> 18
<212> DNA
<213> Em14
<400> 8
gactgcgtac gaattatg 18
<210> 9
<211> 17
<212> DNA
<213> Me8
<400> 9
tgagtccaaa ccggtgc 17
<210> 10
<211> 18
<212> DNA
<213> Em23
<400> 10
gactgcgtac gaatttaa 18
<210> 11
<211> 17
<212> DNA
<213> Me9
<400> 11
tgagtccaaa ccggaac 17
<210> 12
<211> 18
<212> DNA
<213> Em30
<400> 12
gactgcgtac gaatttca 18
<210> 13
<211> 17
<212> DNA
<213> Me10
<400> 13
tgagtccaaa ccggatg 17
<210> 14
<211> 18
<212> DNA
<213> Em36
<400> 14
gactgcgtac gaattgga 18
<210> 15
<211> 17
<212> DNA
<213> Me32
<400> 15
tgagtccaaa ccgggct 17
Claims (7)
1. a kind of SRAP labeling methods with fluorescence labels, it is characterised in that：Its forward primer is the primer with fluorescence labels.
2. labeling method according to claim 1, it is characterised in that：The fluorescence labels are IRDye 700 or IRDye
800。
3. labeling method according to claim 1, it is characterised in that：Step is as follows：
(1) extract the DNA in sample to be checked；
(2) with forward primer be the primer pair with fluorescence labels enter performing PCR amplification；
(3) result detection：DNA cloning result is detected.
4. labeling method according to claim 3, it is characterised in that：In step (2), the system of the amplification is：1×
PCR buffer,Mg2+1.8mmol/L, dNTP 0.15mmol/L, each 0.4 μm of ol/L of forward and reverse primer, DNA profiling 20ng, Taq
Enzyme 1U, reaction system are 10 μ L.
5. labeling method according to claim 3, it is characterised in that：In step (2), the program of the PCR amplifications is：94
DEG C denaturation 5min；94 DEG C of denaturation 40s, 35 DEG C of renaturation 60s, 72 DEG C of extension 60s, totally 5 circulations；94 DEG C of 40s, 50 DEG C of 60s, 72
DEG C 1min, totally 35 circulations；72 DEG C are continued to extend 10min, 4 DEG C of insulations.
6. labeling method according to claim 4, it is characterised in that：In step (3), the method for the detection is polypropylene
Acrylamide gel electrophoresis, silver staining electrophoresis or the method using genetic analysis systems LI-COR4300 electrophoresis.
7. it is a kind of identification wheat breed MY29 and MY29TP method, it is characterised in that：Using shown in SEQ ID NO.3～6
Primer pair 2 shown in primer pair 1 or SEQ ID NO.15～6, is carried out according to claim 1～6 any one methods described
Detection.
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| CN201611256864.5A CN106521015A (en) | 2016-12-30 | 2016-12-30 | SRAP marking method with fluorescent label |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104711348A (en) * | 2015-03-11 | 2015-06-17 | 中蔬种业科技（北京）有限公司 | Molecular marker S9.5 co-separated from spinach gender gene Y and application of molecular marker S9.5 |
-
2016
- 2016-12-30 CN CN201611256864.5A patent/CN106521015A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104711348A (en) * | 2015-03-11 | 2015-06-17 | 中蔬种业科技（北京）有限公司 | Molecular marker S9.5 co-separated from spinach gender gene Y and application of molecular marker S9.5 |
Non-Patent Citations (5)
| Title |
|---|
| JUAN J. RUIZ等: "genetic variability and relationship of closely related spanish traditional cultivars of tomato as detected by SRAP and SSR markers", 《J. AMER. SOC. HORT. SCI》 * |
| LI和QUIROS: "sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica", 《THEOR APPL GENET》 * |
| XIAODONG WANG等: "identification of QTLs associated with oil content in a high-oil brassica napus cultivar and construction of a high-density consensus map for QTLs comparison in B. napus", 《PLOS ONE》 * |
| 吴则东等: "适合甜菜品种鉴定的SRAP核心引物的筛选", 《中国农学通报》 * |
| 杨在君等: "利用SRAP分子标记评价小麦三雌蕊近等基因系的遗传背景", 《核农学报》 * |
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