CN108456720A - A method of based on SRAP Marker Identification butterfly orchid varieties - Google Patents
A method of based on SRAP Marker Identification butterfly orchid varieties Download PDFInfo
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- CN108456720A CN108456720A CN201810289958.5A CN201810289958A CN108456720A CN 108456720 A CN108456720 A CN 108456720A CN 201810289958 A CN201810289958 A CN 201810289958A CN 108456720 A CN108456720 A CN 108456720A
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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Abstract
The invention discloses a kind of methods based on SRAP Marker Identification butterfly orchid varieties, include the following steps：Extract iris genomic DNA；It is combined using SRAP primers, PCR amplification is carried out by template of iris genomic DNA；By the DNA fragmentation after amplification, 14gL is utilized‑1Agarose gel electrophoresis detect SRAP amplified productions；Electrophoresis result is analyzed, and then identifies butterfly orchid variety；Forward primer in the SRAP primers combination is selected from me1 me10 (SEQ ID No.1 10), and reverse primer is selected from em1 em10 (SEQ ID No.11 20).The advantage of the invention is that：Method provided by the invention is simple and efficient disposably by a variety of butterfly orchid variety authentication process；Method provided by the invention, primer dosage are less, it is only necessary to 0.4 μm of olL 1.
Description
Technical field
The invention belongs to agriculture fields, more particularly to a kind of method based on SRAP Marker Identification butterfly orchid varieties.
Background technology
Iris (Phalaenopsis aphrodite Rchb.F.) is orchid family Phalaenopsis, originates in hylaeion hypotropicum
Area, for growing nonparasitically upon another plant property orchid.The coarse aerial root of iris white is exposed at around blade, in addition to the work for absorbing nutrient in air
With outer, also growth and photosynthesis.In the time in the new year, Phalaenopsis plants extract long bennet out from axil, and output shape
Flower as butterfly dances in the air, the deep favor by flower fans are known as the title of " cattleya queen consort ".Type is various, only leans on conventional identification
Method be difficult provide admissible evidence.
That is applied with the development of biotechnology and its in crop breeding deepens continuously, and scientists, which propose to apply, divides
Sub- labelling technique carries out heredity and the authenticity identification of variety of crops.SRAP is easy to operate, using the primer of long 17-18bp with
And 50 DEG C of annealing temperature, it ensure that the stability of result.3 selection bases are held to can be obtained more by changing SRAP primers 3 '
Primer can carry out multiple combinations since upstream and downstream primer can freely be assembled with a small number of primers, greatly reduce
The expense of synthetic primer, while also improving the utilization rate of primer.SRAP is easy to operate, stable amplification result, efficiently, repeatability
The advantages that good, is applied to map construction, comparative genomics, analysis of genetic diversity and demarcates in the research of gene.
So far, although someone applies SRAP labels on iris Genetic relationship, there is not yet
The report of a variety of butterfly orchid variety identifications is disposably carried out using SRAP labels.
Invention content
It cannot be disposably by a variety of iris product technical problem to be solved by the present invention lies in overcoming existing SRAP to mark
Kind identification, and develop a kind of quick, method conveniently and efficiently based on SRAP Marker Identification butterfly orchid varieties.
The object of the present invention is to provide a kind of methods based on SRAP Marker Identification butterfly orchid varieties.
Technical scheme is as follows：
A method of based on SRAP Marker Identification butterfly orchid varieties, which is characterized in that include the following steps：
(1) iris genomic DNA is extracted；
(2) it utilizes SRAP primers to combine, PCR amplification is carried out by template of iris genomic DNA；
(3) by the DNA fragmentation after amplification, 14gL is utilized-1Agarose gel electrophoresis detect SRAP amplified productions；
(4) electrophoresis result is analyzed, and then identifies butterfly orchid variety；
Forward primer in the SRAP primers combination is selected from me1-me10 (SEQ ID No.1-10), and reverse primer is selected from
em1-em10(SEQ ID No.11-20)。
Preferably, the forward primer in the SRAP primers combination is me1 (SEQ ID No.1).
It is further preferred that the reverse primer in the SRAP primers combination is em1 (SEQ ID No.11).
It is further preferred that the reverse primer in the SRAP primers combination is em10 (SEQ ID No.20).
Preferably, the PCR amplification system is made of 20 μ L iris SRAP reaction systems, the iris SRAP reactions
System includes：75ng template DNAs, 0.4 μm of olL-1Primer, 0.6-0.9mmolL-1DNTPs, 1.5U Taq DNA polymerizations
Enzyme, 2.0mmolL-1MgCl2, 2.0 μ L 10 × PCR buffer solutions.
It is further preferred that the dNTPs in the PCR amplification system is 0.75mmolL-1。
The advantage of the invention is that：
1, method provided by the invention is simple and efficient disposably by a variety of butterfly orchid variety authentication process；
2, method provided by the invention, primer dosage are less, it is only necessary to 0.4 μm of olL-1。
Description of the drawings
Fig. 1 is that the SRAP of a preferred embodiment of the invention is composed.
Fig. 2 is that the SRAP of another preferred embodiment of the present invention is composed.
Wherein：M, molecular weight marker；1, mountain breeze scape, 2, Chinese red, 3, big capsicum, 4, firebird, 5,2556,6, radiance, 7,
Fiery phoenix.
Specific implementation mode
With reference to specific embodiment, the present invention is further elaborated, but the present invention is not limited to following embodiments.Under
It is conventional method to state method described in embodiment unless otherwise instructed.The raw material unless otherwise instructed can be from open quotient
Industry approach and obtain.
Embodiment 1
A method of based on SRAP Marker Identification butterfly orchid varieties, include the following steps：
(1) extraction and detection of genomic DNA
The stalwartnesses of 7 kinds of common irises such as selection mountain breeze scape, Chinese red, big capsicum, firebird, 2556, radiance, fiery phoenix,
Disease-free plant (the same minimum 3 plants of mixed samplings of kind) with this variety characteristic, every plant of clip 1-2 piece petal, with changing
Into micro CTAB methods extract DNA.It takes 10 μ L template DNAs to be diluted to 3 000 μ L with 1 × TE, divides on ultraviolet specrophotometer
Not Ce Ding 260nm and 280nm light absorption values (OD), with OD260/OD280 detect each sample purity and calculate its concentration.Use 8g
L-1Agarose gel electrophoresis detects the integrality of DNA.
(2) genomic DNA extension and SRAP analyses
Amplified reaction carries out on the GeneAmp PCR System 9 600 that Applera companies of the U.S. produce.Using 20 μ
L systems：75ng template DNAs, 0.4 μm of olL-1SRAP primers combine, 0.75mmolL-1DNTPs, 1.5U TaqDNA polymerizations
Enzyme, 2.0mmolL-1MgCl2, the ultra-pure water of sterilizing is added to 20 μ L in 2.0 μ L10 × PCR buffer solutions.PCR reaction systems determine
Afterwards, under conditions of keeping reaction system other factors consistent, change monofactor, screen optimal parameter.
Forward primer in the SRAP primers combination is me1 (SEQ ID No.1), anti-in the SRAP primers combination
It is em1 (SEQ ID No.11) to primer.
SRAP reacts thermocycling program to be implemented with reference to the methods of G.Li, i.e. 94 DEG C of pre-degeneration 4min, 94 DEG C of denaturation 1min, and 35
DEG C annealing 1min, 72 DEG C extension 1min, 5 cycle；72 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C of extension 1min, 35 are followed
Ring, 72 DEG C of extension 10min, is finally stored in 4 DEG C of refrigerators.
Pcr amplification product 14gL-1Agarose gel electrophoresis detection, 90V voltage stabilizing electrophoresis 1h, EB dyeing, in UVP public affairs
It observes and takes pictures on 000 gel imaging systems of GDS8 of department.
As shown in Figure 1, being combined using above-mentioned SRAP primers, SRAP spectrums have polymorphism, can be by 7 butterfly orchid varieties
Disposably distinguish.
Embodiment 2
A method of based on SRAP Marker Identification butterfly orchid varieties, include the following steps：
(1) extraction and detection of genomic DNA
The stalwartnesses of 7 kinds of common irises such as selection mountain breeze scape, Chinese red, big capsicum, firebird, 2556, radiance, fiery phoenix,
Disease-free plant (the same minimum 3 plants of mixed samplings of kind) with this variety characteristic, every plant of clip 1-2 piece petal, with changing
Into micro CTAB methods extract DNA.It takes 10 μ L template DNAs to be diluted to 3 000 μ L with 1 × TE, divides on ultraviolet specrophotometer
Not Ce Ding 260nm and 280nm light absorption values (OD), with OD260/OD280 detect each sample purity and calculate its concentration.Use 8g
L-1Agarose gel electrophoresis detects the integrality of DNA.
(2) genomic DNA extension and SRAP analyses
Amplified reaction carries out on the GeneAmp PCR System 9 600 that Applera companies of the U.S. produce.Using 20 μ
L systems：75ng template DNAs, 0.4 μm of olL-1SRAP primers combine, 0.75mmolL-1DNTPs, 1.5U TaqDNA polymerizations
Enzyme, 2.0mmolL-1MgCl2, the ultra-pure water of sterilizing is added to 20 μ L in 2.0 μ L10 × PCR buffer solutions.PCR reaction systems determine
Afterwards, under conditions of keeping reaction system other factors consistent, change monofactor, screen optimal parameter.
Forward primer in the SRAP primers combination is me1 (SEQ ID No.1), anti-in the SRAP primers combination
It is em10 (SEQ ID No.20) to primer.
SRAP reacts thermocycling program to be implemented with reference to the methods of G.Li, i.e. 94 DEG C of pre-degeneration 4min, 94 DEG C of denaturation 1min, and 35
DEG C annealing 1min, 72 DEG C extension 1min, 5 cycle；72 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C of extension 1min, 35 are followed
Ring, 72 DEG C of extension 10min, is finally stored in 4 DEG C of refrigerators.
Pcr amplification product 14gL-1Agarose gel electrophoresis detection, 90V voltage stabilizing electrophoresis 1h, EB dyeing, in UVP public affairs
It observes and takes pictures on 000 gel imaging systems of GDS8 of department.
As shown in Fig. 2, being combined using above-mentioned SRAP primers, SRAP spectrums have polymorphism, can be by 7 butterfly orchid varieties
Disposably distinguish.
Embodiment 3
Difference lies in the dNTPs is 0.6mmolL to the present embodiment with embodiment 1-1。
Embodiment 4
Difference lies in the dNTPs is 0.9mmolL to the present embodiment with embodiment 1-1。
Embodiment 5
Difference lies in the forward primer in the SRAP primers combination is me2 (SEQ ID to the present embodiment with embodiment 1
No.2), the reverse primer in the SRAP primers combination is em2 (SEQ ID No.12).
Embodiment 6
Difference lies in the forward primer in the SRAP primers combination is me3 (SEQ ID to the present embodiment with embodiment 1
No.3), the reverse primer in the SRAP primers combination is em3 (SEQ ID No.13).
Embodiment 7
Difference lies in the forward primer in the SRAP primers combination is me4 (SEQ ID to the present embodiment with embodiment 1
No.4), the reverse primer in the SRAP primers combination is em4 (SEQ ID No.14).
Embodiment 8
Difference lies in the forward primer in the SRAP primers combination is me5 (SEQ ID to the present embodiment with embodiment 1
No.5), the reverse primer in the SRAP primers combination is em5 (SEQ ID No.15).
Embodiment 9
Difference lies in the forward primer in the SRAP primers combination is me6 (SEQ ID to the present embodiment with embodiment 1
No.6), the reverse primer in the SRAP primers combination is em6 (SEQ ID No.16).
Embodiment 10
Difference lies in the forward primer in the SRAP primers combination is me7 (SEQ ID to the present embodiment with embodiment 1
No.7), the reverse primer in the SRAP primers combination is em7 (SEQ ID No.17).
Embodiment 11
Difference lies in the forward primer in the SRAP primers combination is me8 (SEQ ID to the present embodiment with embodiment 1
No.8), the reverse primer in the SRAP primers combination is em8 (SEQ ID No.18).
Embodiment 12
Difference lies in the forward primer in the SRAP primers combination is me9 (SEQ ID to the present embodiment with embodiment 1
No.9), the reverse primer in the SRAP primers combination is em9 (SEQ ID No.19).
Embodiment 13
Difference lies in the forward primer in the SRAP primers combination is me10 (SEQ ID to the present embodiment with embodiment 1
No.10), the reverse primer in the SRAP primers combination is em10 (SEQ ID No.20).
Embodiment 14
Difference lies in the forward primer in the SRAP primers combination is me10 (SEQ ID to the present embodiment with embodiment 1
No.10, the reverse primer in the SRAP primers combination is em1 (SEQ ID No.11).
Above-mentioned specific implementation mode is only explained in detail technical scheme of the present invention, the present invention not only only office
Be limited to above-described embodiment, it will be understood by those skilled in the art that it is every according to above-mentioned principle and spirit on the basis of the present invention
It improves, substitute, it all should be within protection scope of the present invention.
Sequence table
<110>Fuqing Branch of Fujian Normal University
<120>A method of based on SRAP Marker Identification butterfly orchid varieties
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 1
tgagtccaaa ccggata 17
<210> 2
<211> 17
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 2
tgagtccaaa ccggagc 17
<210> 3
<211> 17
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 3
tgagtccaaa ccggaat 17
<210> 4
<211> 17
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 4
tgagtccaaa ccggacc 17
<210> 5
<211> 17
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 5
tgagtccaaa ccggaag 17
<210> 6
<211> 17
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 6
tgagtccaaa ccggtaa 17
<210> 7
<211> 17
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 7
tgagtccaaa ccggacc 17
<210> 8
<211> 17
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 8
tgagtccaaa ccggtgc 17
<210> 9
<211> 17
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 9
tgagtccaaa ccgggct 17
<210> 10
<211> 17
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 10
tgagtccaaa ccggtaa 17
<210> 11
<211> 18
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 11
gactgggtac gaattaat 18
<210> 12
<211> 18
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 12
gactgggtac gaatttgc 18
<210> 13
<211> 18
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 13
gactgggtac gaattgac 18
<210> 14
<211> 18
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 14
gactgggtac gaatttga 18
<210> 15
<211> 18
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 15
gactgggtac gaattaac 18
<210> 16
<211> 18
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 16
gactgggtac gaattgca 18
<210> 17
<211> 18
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 17
gactgggtac gaattcaa 18
<210> 18
<211> 18
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 18
gactgggtac gaattcga 18
<210> 19
<211> 18
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 19
gactgggtac gaattcac 18
<210> 20
<211> 18
<212> DNA
<213>Artificial sequence (Phalaenopsis aphrodite Rchb. F.)
<400> 20
gactgggtac gaattcca 18
Claims (6)
1. a kind of method based on SRAP Marker Identification butterfly orchid varieties, which is characterized in that include the following steps：
(1) iris genomic DNA is extracted；
(2) it utilizes SRAP primers to combine, PCR amplification is carried out by template of iris genomic DNA；
(3) by the DNA fragmentation after amplification, 14gL is utilized-1Agarose gel electrophoresis detect SRAP amplified productions；
(4) electrophoresis result is analyzed, and then identifies butterfly orchid variety；
Forward primer in the SRAP primers combination is selected from me1-me10 (SEQ ID No.1-10), and reverse primer is selected from em1-
em10(SEQ ID No.11-20)。
2. according to the method described in claim 1, it is characterized in that：Forward primer in the SRAP primers combination is me1
(SEQ ID No.1)。
3. method according to claim 1 or 2, it is characterised in that：Reverse primer in the SRAP primers combination is em1
(SEQ ID No.11)。
4. method according to claim 1 or 2, it is characterised in that：Reverse primer in SRAP primers combination is
em10(SEQ ID No.20)。
5. according to the method described in claim 1, it is characterized in that：The PCR amplification system is by 20 μ L irises SRAP reactions
System is constituted, and the iris SRAP reaction systems include：75ng template DNAs, 0.4 μm of olL-1Primer, 0.6-0.9mmol
L-1DNTPs, 1.5U Taq archaeal dna polymerases, 2.0mmolL-1MgCl2, 2.0 μ L 10 × PCR buffer solutions.
6. according to the method described in claim 5, it is characterized in that：DNTPs in the PCR amplification system is 0.75mmol
L-1。
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| CN201810289958.5A CN108456720A (en) | 2018-04-03 | 2018-04-03 | A method of based on SRAP Marker Identification butterfly orchid varieties |
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Cited By (2)
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| CN112695124A (en) * | 2021-01-29 | 2021-04-23 | 广东省农业科学院环境园艺研究所 | Phalaenopsis SSR molecular marker primer composition and application thereof |
| CN114085921A (en) * | 2021-10-27 | 2022-02-25 | 海南省农业科学院热带园艺研究所 | Genetic marker of phalaenopsis based on RTE (terminal-to-terminal insertion deletion) polymorphism and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112695124A (en) * | 2021-01-29 | 2021-04-23 | 广东省农业科学院环境园艺研究所 | Phalaenopsis SSR molecular marker primer composition and application thereof |
| CN114085921A (en) * | 2021-10-27 | 2022-02-25 | 海南省农业科学院热带园艺研究所 | Genetic marker of phalaenopsis based on RTE (terminal-to-terminal insertion deletion) polymorphism and application |
| CN114085921B (en) * | 2021-10-27 | 2023-10-24 | 海南省农业科学院热带园艺研究所 | Genetic marker of phalaenopsis based on RTE indel polymorphism and application |
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