CN102154479A - Primer sequence for detecting purity of Jinyou 303 cucumber hybrid seeds and detection method thereof - Google Patents
Primer sequence for detecting purity of Jinyou 303 cucumber hybrid seeds and detection method thereof Download PDFInfo
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- CN102154479A CN102154479A CN2011100333006A CN201110033300A CN102154479A CN 102154479 A CN102154479 A CN 102154479A CN 2011100333006 A CN2011100333006 A CN 2011100333006A CN 201110033300 A CN201110033300 A CN 201110033300A CN 102154479 A CN102154479 A CN 102154479A
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Abstract
The invention relates to a primer sequence for detecting the purity of Jinyou 303 cucumber hybrid seeds and a detection method thereof. The primer sequence is composed of an upstream primer and a downstream primer, wherein the upstream primer is a nucleotide sequence as shown in SEQ ID NO.1 in a sequence table, and the downstream primer is a nucleotide sequence as shown in SEQ ID NO.2 in the sequence table. The primer sequence has the advantages of low cost and the like, is quick, simple, stable and reliable and is not affected by environment conditions, and the purity of one batch of 303 cucumber hybrid seeds can be identified in 5-6 hours only. A great quantity of manpower and soil fertility can be saved, and an identification result is quick and accurate. The primer sequence and the detection method in the invention have good application value on identifying the purity of cucumber seeds.
Description
Technical field
The present invention relates to biological technical field, relate to a kind of primer sequence and detection method that is used to detect the excellent No. 303 sub-purity of cucumber hybrid in Tianjin particularly.
Background technology
Seed purity identifies it is the key link that guarantees seed quality.Along with the quickening of breeding process, new breeds of cucumbers quantity constantly increases, and the quantity of material that need carry out the variety evaluation will be increasing.Conventional in the past purity identifies it all is to carry out in the field, the knot melon phase is by feature and the consistence of expert to the field observation kind, there is long, problem such as workload big, be subject to Effect of Environmental, phenotypic characteristic has to a certain degree deviation of cycle, may influence the accuracy that variety is identified, and directly have influence on the popularization of breeding; Because qualification cycle is long, and the seed of current year's production often will arrive next year and could sell, cause the business opportunity forfeiture in addition.
The birth of molecular marking technique is for the Molecular Identification of seed purity has been established technical foundation.Molecule marker can all can detect in each tissue, each etap of organism directly with the form performance of DNA, is not subjected to season, environmental restraint, does not exist expression whether to wait problem.Especially little satellite (SSR) technology, microsatellite DNA quantity is many and be evenly distributed in the genome, has abundant polymorphism, and microsatellite locus detects and can show homozygote and heterozygote, easily detection, good reproducibility, save time, be suitable for big flux distribution.The SSR authentication method of cucumber seeds purity does not appear in the newspapers as yet at present.
Summary of the invention
The purpose of this invention is to provide a kind of primer sequence that is used to detect the excellent No. 303 sub-purity of cucumber hybrid in Tianjin.
Another object of the present invention is to overcome the deficiencies in the prior art, and a kind of method that can detect the excellent No. 303 sub-purity of cucumber hybrid in Tianjin apace is provided.
Technical scheme of the present invention is summarized as follows:
Be used to detect the primer sequence of the excellent No. 303 sub-purity of cucumber hybrid in Tianjin, it is made up of upstream primer and downstream primer, described upstream primer is the described nucleotide sequence of SEQ ID NO.1 in the sequence table, and described downstream primer is the described nucleotide sequence of SEQ ID NO.2 in the sequence table.
Detect the method for the excellent No. 303 sub-purity of cucumber hybrid in Tianjin, comprise the steps:
(1) extracts the excellent No. 303 sub-genomic dnas of cucumber hybrid in Tianjin;
(2) carry out pcr amplification: in the special-purpose thin-walled tube of pcr amplification, add the sub-genome DNA10～20ng of excellent No. 303 cucumber hybrids in Tianjin, add again in the sequence table the described nucleotide sequence 15～30ng of SEQ ID NO.2 in the described nucleotide sequence 15～30ng of SEQ ID NO.1, the sequence table, 1 times contain Mg
2+PCR damping fluid, dNTP 2mmol, Taq archaeal dna polymerase 0.5～1 unit, add aseptic double distilled water to 10 μ l; The special-purpose thin-walled tube of pcr amplification is put into the PCR instrument increase, amplification condition is: 94 ℃ of pre-sex change 180 seconds; 94 ℃ of sex change 30 seconds, 50-55 ℃ of annealing 30 seconds, 72 ℃ were extended 30 circulations 60 seconds; 72 ℃ are extended 7min again, and amplification is finished;
(3) gel electrophoresis analysis of pcr amplification product: adopt 6% denaturing polyacrylamide gel to carry out electrophoretic separation amplified production; Add the denaturant gel sample-loading buffer in amplified production, be splined on the sex change polypropylene amine gel of 30 minutes prerunnings, the permanent power electrophoresis of 70W to bromjophenol blue has just arrived the other end of gel, and gel is through observing under visible light behind the cma staining, taking a picture;
(4) according to the relative position of being identified sample band on gel, detect the excellent No. 303 sub-purity of cucumber hybrid in Tianjin.
That the present invention has is quick, easy, stable, reliable, low-cost, be not subjected to advantages such as environmental influence, and the excellent No. 303 sub-purity of cucumber hybrid in Tianjin are identified only needs just can finish in 5-6 hour one batch evaluation.Saved a large amount of manpowers, soil fertility, qualification result quick and precisely.On identifying, cucumber seeds purity has very big using value.
Description of drawings
Fig. 1 is the gel electrophoresis figure of the excellent No. 303 cucumber pcr amplification products in Tianjin.
Embodiment
The present invention is further illustrated below in conjunction with drawings and the specific embodiments, and the following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.
Embodiment 1
Detect the method for the excellent No. 303 sub-purity of cucumber hybrid in Tianjin, comprise the steps:
(1) genomic dna of the tip of a root after excellent No. 303 cucumber hybrids in extraction Tianjin germinateed 35 hours;
(2) carry out pcr amplification: in the special-purpose thin-walled tube of pcr amplification, add the sub-genome DNA10ng of excellent No. 303 cucumber hybrids in Tianjin, add again in the sequence table the described nucleotide sequence 15ng of SEQ ID NO.2 in the described nucleotide sequence 15ng of SEQ ID NO.1, the sequence table, 1 times contain Mg
2+PCR damping fluid, dNTP 2mmol, Taq archaeal dna polymerase 0.5 unit, add aseptic double distilled water to 10 μ l; The special-purpose thin-walled tube of pcr amplification is put into the PCR instrument increase, amplification condition is: 94 ℃ of pre-sex change 180 seconds; 94 ℃ of sex change 30 seconds, 50 ℃ of annealing 30 seconds, 72 ℃ were extended 30 circulations 60 seconds; 72 ℃ are extended 7min again, and amplification is finished;
(3) gel electrophoresis analysis of pcr amplification product: adopt 6% denaturing polyacrylamide gel to carry out electrophoretic separation amplified production; Add the denaturant gel sample-loading buffer in amplified production, be splined on the sex change polypropylene amine gel of 30 minutes prerunnings, the permanent power electrophoresis of 70W to bromjophenol blue has just arrived the other end of gel, and gel is through observing under visible light behind the cma staining, taking a picture;
(4) according to the relative position of being identified sample band on gel, detect the excellent No. 303 sub-purity of cucumber hybrid in Tianjin.As shown in Figure 1, excellent No. 303 cenospeciess in Tianjin have the band shown in arrow 1 and the arrow 2 simultaneously at about 156bp place, have only 1 or 2 in this position, and perhaps the two does not all have, and is not this kind.
Detect the method for the excellent No. 303 sub-purity of cucumber hybrid in Tianjin, comprise the steps:
(1) extracts the excellent No. 303 sub-genomic dnas of cucumber hybrid in Tianjin;
(2) carry out pcr amplification: in the special-purpose thin-walled tube of pcr amplification, add the sub-genome DNA20ng of excellent No. 303 cucumber hybrids in Tianjin, add again in the sequence table the described nucleotide sequence 30ng of SEQ ID NO.2 in the described nucleotide sequence 30ng of SEQ ID NO.1, the sequence table, 1 times contain Mg
2+PCR damping fluid, dNTP 2mmol, Taq archaeal dna polymerase 1 unit, add aseptic double distilled water to 10 μ l; The special-purpose thin-walled tube of pcr amplification is put into the PCR instrument increase, amplification condition is: 94 ℃ of pre-sex change 180 seconds; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 30 circulations 60 seconds; 72 ℃ are extended 7min again, and amplification is finished;
(3) gel electrophoresis analysis of pcr amplification product: adopt 6% denaturing polyacrylamide gel to carry out electrophoretic separation amplified production; Add the denaturant gel sample-loading buffer in amplified production, be splined on the sex change polypropylene amine gel of 30 minutes prerunnings, the permanent power electrophoresis of 70W to bromjophenol blue has just arrived the other end of gel, and gel is through observing under visible light behind the cma staining, taking a picture;
(4) according to the relative position of being identified sample band on gel, detect the excellent No. 303 sub-purity of cucumber hybrid in Tianjin.
Embodiment 3
Detect the method for the excellent No. 303 sub-purity of cucumber hybrid in Tianjin, comprise the steps:
(1) extracts the excellent No. 303 sub-genomic dnas of cucumber hybrid in Tianjin;
(2) carry out pcr amplification: in the special-purpose thin-walled tube of pcr amplification, add the sub-genome DNA15ng of excellent No. 303 cucumber hybrids in Tianjin, add again in the sequence table the described nucleotide sequence 20ng of SEQ ID NO.2 in the described nucleotide sequence 20ng of SEQ ID NO.1, the sequence table, 1 times contain Mg
2+PCR damping fluid, dNTP 2mmol, Taq archaeal dna polymerase 0.8 unit, add aseptic double distilled water to 10 μ l; The special-purpose thin-walled tube of pcr amplification is put into the PCR instrument increase, amplification condition is: 94 ℃ of pre-sex change 180 seconds; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 60 seconds, and 30 are followed bad; 72 ℃ are extended 7min again, and amplification is finished;
(3) gel electrophoresis analysis of pcr amplification product: adopt 6% denaturing polyacrylamide gel to carry out electrophoretic separation amplified production; Add the denaturant gel sample-loading buffer in amplified production, be splined on the sex change polypropylene amine gel of 30 minutes prerunnings, the permanent power electrophoresis of 70W to bromjophenol blue has just arrived the other end of gel, and gel is through observing under visible light behind the cma staining, taking a picture;
(4) according to the relative position of being identified sample band on gel, detect the excellent No. 303 sub-purity of cucumber hybrid in Tianjin.
Excellent No. 303 cucumber hybrid in Tianjin are sold by Tianjin Kerun Agricultural Science ﹠ Technology Co., Ltd..
Claims (2)
1. be used to detect the primer sequence of the excellent No. 303 sub-purity of cucumber hybrid in Tianjin, it is characterized in that it is made up of upstream primer and downstream primer, described upstream primer is the described nucleotide sequence of SEQ ID NO.1 in the sequence table, and described downstream primer is the described nucleotide sequence of SEQ ID NO.2.
2. detect the method for the excellent No. 303 sub-purity of cucumber hybrid in Tianjin, it is characterized in that comprising the steps:
(1) extracts the excellent No. 303 sub-genomic dnas of cucumber hybrid in Tianjin;
(2) carry out pcr amplification: in the special-purpose thin-walled tube of pcr amplification, add the sub-genome DNA10～20ng of excellent No. 303 cucumber hybrids in Tianjin, add again in the sequence table the described nucleotide sequence 15～30ng of SEQ ID NO.2 in the described nucleotide sequence 15～30ng of SEQ ID NO.1, the sequence table, 1 times contain Mg
2+PCR damping fluid, dNTP 2mmol, Taq archaeal dna polymerase 0.5～1 unit, add aseptic double distilled water to 10 μ l; The special-purpose thin-walled tube of pcr amplification is put into the PCR instrument increase, amplification condition is: 94 ℃ of pre-sex change 180 seconds; 94 ℃ of sex change 30 seconds, 50-55 ℃ of annealing 30 seconds, 72 ℃ were extended 30 circulations 60 seconds; 72 ℃ are extended 7min again, and amplification is finished;
(3) gel electrophoresis analysis of pcr amplification product: adopt 6% denaturing polyacrylamide gel to carry out electrophoretic separation amplified production; Add the denaturant gel sample-loading buffer in amplified production, be splined on the sex change polypropylene amine gel of 30 minutes prerunnings, the permanent power electrophoresis of 70W to bromjophenol blue has just arrived the other end of gel, and gel is through observing under visible light behind the cma staining, taking a picture;
(4) according to the relative position of being identified sample band on gel, detect the excellent No. 303 sub-purity of cucumber hybrid in Tianjin.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361425A (en) * | 2013-06-13 | 2013-10-23 | 广东省农业科学院蔬菜研究所 | Primer and method for identifying purity of cucumber 'Yuexiu No.3' hybrid seed |
| CN104726607A (en) * | 2015-04-13 | 2015-06-24 | 天津科润农业科技股份有限公司 | Nucleotide sequence and detecting method for detecting Tianjin optimal No. 406 cucumber hybrid seed purity |
| CN104762394A (en) * | 2015-04-13 | 2015-07-08 | 天津科润农业科技股份有限公司 | Nucleotide sequence and detection method for detecting purity of Jinyou #401 cucumber hybrid seed |
-
2011
- 2011-01-31 CN CN2011100333006A patent/CN102154479A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361425A (en) * | 2013-06-13 | 2013-10-23 | 广东省农业科学院蔬菜研究所 | Primer and method for identifying purity of cucumber 'Yuexiu No.3' hybrid seed |
| CN103361425B (en) * | 2013-06-13 | 2015-06-17 | 广东省农业科学院蔬菜研究所 | Primer and method for identifying purity of cucumber 'Yuexiu No.3' hybrid seed |
| CN104726607A (en) * | 2015-04-13 | 2015-06-24 | 天津科润农业科技股份有限公司 | Nucleotide sequence and detecting method for detecting Tianjin optimal No. 406 cucumber hybrid seed purity |
| CN104762394A (en) * | 2015-04-13 | 2015-07-08 | 天津科润农业科技股份有限公司 | Nucleotide sequence and detection method for detecting purity of Jinyou #401 cucumber hybrid seed |
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Application publication date: 20110817 |