CN102533946A - Molecular marker identification method of cucumber branchiness related gene - Google Patents
Molecular marker identification method of cucumber branchiness related gene Download PDFInfo
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- CN102533946A CN102533946A CN201010590382XA CN201010590382A CN102533946A CN 102533946 A CN102533946 A CN 102533946A CN 201010590382X A CN201010590382X A CN 201010590382XA CN 201010590382 A CN201010590382 A CN 201010590382A CN 102533946 A CN102533946 A CN 102533946A
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Abstract
The invention relates to a molecular marker identification method of cucumber branchiness related gene. The method comprises the steps of: carrying out PCR (polymerase chain reaction) amplification with DNA (deoxyribonucleic acid) of a cucumber variety or line with unknown branchiness and with CSWCT17 as a primer, carrying out 3% agarose gel electrophoresis detection on the amplification product or carrying out 7.5% denaturalized polyacrylamide gel electrophoresis detection on the amplification product, and judging the cucumber variety or line with unknown branchiness to be a variety or line with weak branchiness if the amplified band form is consistent with the band form of cucumber Dongnong 803 with weak branchiness, wherein the PCR amplification process comprises the steps of: pre-denaturizing at 94 DEG C for 5 minutes, denaturalizing at 94 DEG C for 30 seconds, annealing at 67 DEG C for 1 minute, circulating 35 times, drawing at 72 DEG C for 1 minute, drawing at 72 DEG C for 10 minutes, and finally storing at 4 DEG C. The molecular marker identification method provided by the invention is used for identifying the cucumber branchiness related gene.
Description
Technical field:
The present invention relates to a kind of molecular marker identification method of cucumber branchiness genes involved, belong to plant biotechnology field.
Background technology:
Branched growing way is one of important character of cucumber plant type.Can grow branch on the cucumber major branch, the branch of can also regenerating on the branch.What are relevant with kind and cultivation condition for the branch amount purpose, and generally the strong middle-late ripening variety of growth potential is more than early maturing variety.The length of stem and branched what etc. characteristic and habit often be the foundation of cucumber plant adjustment, the thickness of stem and the length of internode then are one of diagnosis plant power and output sign just.Plant is thin and delicate, bristle is undeveloped, is difficult to obtain high yield; Stem is climing too sturdy, belongs to the surplus of nourishing and growing, and also can influence reproductive growth.The cucumber branch is the important character of cucumber plant type, and its what and power directly affect cultivation condition, is main because how general cultivated cucumber ties melon with major branch.Branch is many will to bring very big inconvenience to the cultivation of cucumber by force with the branch growing way; The grower just often the field interrupt; So not only can bring a lot of unnecessary labours to the cultivation of cucumber; And the plant wound of interrupting can infect disease, improve cost will for the cultivation of cucumber in the middle of invisible.In cucumber cultivation, branch too much can be fought for the space of cucumber plant growth, and just necessary rational close planting is so that field management; The soil is caused very big waste; And branch can fight for nutrition with major branch, if the branch growing way is strong, will influence the major branch growth; Therefore influence the quality and the output of cucumber fruits, tend to relatively to utilize in China that branch is few, the strain a little less than the growing way.Still unclear for the strong and weak molecule mechanism of cucumber branchiness at present, therefore seek and the closely linked molecule marker of cucumber branchiness genes involved, have important application value for accelerating the breed cucumber process.
Summary of the invention:
The objective of the invention is to provide a kind of molecular marker identification method of cucumber branchiness genes involved to the problem of above-mentioned existence; Can utilize above-mentioned cucumber branchiness genes involved molecule marker better, and cucumber branchiness genes involved molecule marker is applied to the assisted Selection of breed cucumber.
Above-mentioned purpose realizes through following technical scheme:
The molecular marker identification method of cucumber branchiness genes involved, this method comprises the steps:
With the cucumber variety of branchiness the unknown or the DNA of strain is template, is that primer carries out pcr amplification with CSWCT17, amplified production is carried out the detection of 3% agarose gel electrophoresis perhaps amplified production is carried out 7.5% denaturing polyacrylamide gel electrophoresis detection; If the banding pattern that amplification obtains is consistent with agricultural 803 banding patterns in known weak branchiness cucumber east, then unknown cucumber variety or the strain of this branchiness is weak branchiness kind or strain, and the program of said pcr amplification is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 56 ℃ of annealing 1min, 35 circulations; 72 ℃ are extended 1min; 72 ℃ are extended 10min, last 4 ℃ of preservations, and described CSWCT17 is:
Primer 1:5 '-TTGAATTATGGGTTCATTTTT-3 ',
Primer 2: 5 '-GACAATGATAAACTTCCCTGA-3 '.
The molecular marker identification method of described cucumber branchiness genes involved; In the said pcr amplification; The PCR reaction system of per 20 μ 1 is following: 10 * buffer:2 μ l, 2mM dNTP:2 μ l, the Primer 1:0.8 μ l of 10pmol/ μ l; The Primer 2:0.8 μ l of 10pmol/ μ l, 25mM MgCl
2: 2.5 μ l, the TaqDNA polysaccharase of 5U/ μ l: 0.2 μ l, the template DNA of 60ng/ μ l: 1 μ l, ddH2O:10.7 μ l.
The application of the molecular marker identification method of cucumber branchiness genes involved in assisted Selection breed cucumber material.
Beneficial effect:
1. the present invention directly with the form performance of DNA, all can detect in each tissue, each etap of organism, does not receive season, environmental restraint; Do not exist expression whether to wait problem, therefore, the present invention can overcome the evaluation of cucumber branchiness and be prone to shortcoming affected by environment; Just can identify the branchiness of cucumber variety and strain and screen in seedling stage through molecule marker; Eliminate strong branchiness plant, reduce man power and material's waste, improve breeding efficiency.Simultaneously, the dna molecular marker technological operation is simple, and is with low cost, and the time spent is short, in one day, can accomplish whole testing processes.
2. required sample size is few, operates easylier, and efficient is high, and is with low cost.
3. the present invention is easy to grasp, and does not receive environmental influence, and repeatability is high.
Description of drawings:
Fig. 1 cucumber branchiness genes involved Markers for Detection result (east farming 804).M:DL2000 DNAMarker; 1: contrast east farming 803; 2～8: agricultural 804 individual plants in east.
The detected result of Fig. 2 cucumber branchiness genes involved molecule marker (east farming 805).M:DL2000 DNAMarker; 1: contrast east farming 803; 2～10: agricultural 804 individual plants in east.
Embodiment:
Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.All primers are synthetic to be accomplished by Sangon Biotech (Shanghai) Co., Ltd..The cucumber material that all are used in following examples is public.
Embodiment 1:
The molecular marker identification method of cucumber branchiness genes involved, this method comprises the steps:
With the cucumber variety of branchiness the unknown or the DNA of strain is template, is that primer carries out pcr amplification with CSWCT17, amplified production is carried out the detection of 3% agarose gel electrophoresis perhaps amplified production is carried out 7.5% denaturing polyacrylamide gel electrophoresis detection; If the banding pattern that amplification obtains is consistent with agricultural 803 banding patterns in known weak branchiness cucumber east, then unknown cucumber variety or the strain of this branchiness is weak branchiness kind or strain, and the program of said pcr amplification is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 56 ℃ of annealing 1min, 35 circulations; 72 ℃ are extended 1min; 72 ℃ are extended 10min, last 4 ℃ of preservations, and described CSWCT17 is:
Primer 1:5 '-TTGAATTATGGGTTCATTTTT-3 ',
Primer 2: 5 '-GACAATGATAAACTTCCCTGA-3 '.
Embodiment 2:
The molecular marker identification method of described cucumber branchiness genes involved; In the said pcr amplification; The PCR reaction system of per 20 μ l is following: 10 * buffer:2 μ l, 2mM dNTP:2 μ l, the Primer 1:0.8 μ l of 10pmol/ μ l; The Primer 2:0.8 μ l of 10pmol/ μ l, 25mM MgCl
2: 2.5 μ l, the TaqDNA polysaccharase of 5U/ μ l: 0.2 μ l, the template DNA of 60ng/ μ l: 1 μ l, ddH2O:10.7 μ l.
Embodiment 3:
The application of the molecular marker identification method of cucumber branchiness genes involved in assisted Selection breed cucumber material.
The cucumber hybrid new breed east farming of cultivating with cucumber seminar of gardening institute of Northeast Agricultural University 804 is a material with Dong Nong 805; Extracting its single plant DNA is template; With 1 cucumber branchiness molecule marker CSWCT17 provided by the invention is that primer carries out pcr amplification; The result shows; The pcr amplification banding pattern of east farming 804 and Dong Nong 805 individual plants is in full accord with weak branchiness contrast east farming 803 amplification banding patterns, and the identical rate that PCR detected result and field branchiness are identified is 100%, has proved that CSWCT17 is the molecule marker of the cucumber branchiness detection of a practicality.
Claims (3)
1. the molecular marker identification method of a cucumber branchiness genes involved is characterized in that: this method comprises the steps: that with the unknown cucumber variety of branchiness or the DNA of strain be template, is that primer carries out pcr amplification with CSWCT17; Amplified production is carried out the detection of 3% agarose gel electrophoresis or amplified production is carried out 7.5% denaturing polyacrylamide gel electrophoresis detection, if the banding pattern that amplification obtains is consistent with eastern agricultural 803 banding patterns of known weak branchiness cucumber, then unknown cucumber variety or the strain of this branchiness is weak branchiness kind or strain; The program of said pcr amplification is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 56 ℃ of annealing 1min, 35 circulations; 72 ℃ are extended 1min; 72 ℃ are extended 10min, last 4 ℃ of preservations, and described CSWCT17 is:
Primer 1:5 '-TTGAATTATGGGTTCATTTTT-3 ',
Primer 2: 5 '-GACAATGATAAACTTCCCTGA-3 '.
2. the molecular marker identification method of cucumber branchiness genes involved according to claim 1; It is characterized in that: in the said pcr amplification; The PCR reaction system of per 20 μ l is following: 10 * buffer:2 μ l, 2mMdNTP:2 μ l, the Primer 1:0.8 μ l of 10pmol/ μ l; The Primer 2:0.8 μ l of 10pmol/ μ l, 25mM MgCl
2: 2.5 μ l, the TaqDNA polysaccharase of 5U/ μ l: 0.2 μ l, the template DNA of 60ng/ μ l: 1 μ l, ddH20:10.7 μ l.
3. the application of the molecular marker identification method of an above-mentioned cucumber branchiness genes involved in assisted Selection breed cucumber material.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232996A (en) * | 2013-04-09 | 2013-08-07 | 南京农业大学 | Chrysanthemum-branching-trait-related molecular marker acquisition method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009086850A1 (en) * | 2008-01-10 | 2009-07-16 | Enza Zaden Beheer B.V. | Marker genetically linked to tobamovirus resistance in cucumber and the use thereof |
| CN101591708A (en) * | 2009-06-11 | 2009-12-02 | 上海交通大学 | cucumber SNP marker and detection method thereof |
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2010
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009086850A1 (en) * | 2008-01-10 | 2009-07-16 | Enza Zaden Beheer B.V. | Marker genetically linked to tobamovirus resistance in cucumber and the use thereof |
| CN101591708A (en) * | 2009-06-11 | 2009-12-02 | 上海交通大学 | cucumber SNP marker and detection method thereof |
Non-Patent Citations (3)
| Title |
|---|
| G. FAZIO ET.AL.,: "Comparative analysis of response to phenotypic and marker-assisted selection for multiple lateral branching in cucumber (Cucumis sativus L.)", 《THEOR APPL GENET》 * |
| GENNARO FAZIO ET.AL.,: "Development and characterization of PCR markers in cucumber", 《J.AMER.SOC.HORT.SCI.》 * |
| 李开盛等: "黄瓜分枝性遗传分析", 《东北农业大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232996A (en) * | 2013-04-09 | 2013-08-07 | 南京农业大学 | Chrysanthemum-branching-trait-related molecular marker acquisition method |
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Application publication date: 20120704 |