CN102251042B - Method for quickly detecting purity of seeds of bottle gourd varieties and kit used by same - Google Patents
Method for quickly detecting purity of seeds of bottle gourd varieties and kit used by same Download PDFInfo
- Publication number
- CN102251042B CN102251042B CN 201110207456 CN201110207456A CN102251042B CN 102251042 B CN102251042 B CN 102251042B CN 201110207456 CN201110207456 CN 201110207456 CN 201110207456 A CN201110207456 A CN 201110207456A CN 102251042 B CN102251042 B CN 102251042B
- Authority
- CN
- China
- Prior art keywords
- dna
- bottle gourd
- ssr
- primer
- seed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Abstract
The invention discloses a method for quickly detecting the purity of the seeds of bottle gourd varieties and a kit used by the same. The method comprises the following steps: (I) partial sequencing of bottle gourd genomic DNA and simple sequence repeat (SSR) primer development and design; (II) calculation of the polymorphism information content (PIC) of SSR primers and screening of combination of diagnostic SSR primers; (III) preparation of standardized DNA fingerprint of the five main cultivated bottle gourd varieties; and (IV) detection of bottle gourd samples to be detected. In the method, because of the use of the 8 pairs of diagnostic SSR primers which are designed independently, the capacity of identifying similar varieties is increased, the efficiency of detection is improved, the detection time is reduced to 4 to 5 hours from 50 to 60 hours which are required by traditional phenotype identification, the detection cost is lowered to about 1/6 of that required by the conventional method, and the accuracy of identification is improved. The method can be promoted and used in seed breeding and seed enterprises.
Description
Technical field
The invention belongs to the crop biological technical field, especially belong to the method and the test kit thereof that utilize diagnostic SSR molecule marker combination rapid detection bottle gourd principal item seed purity.
Background technology
Bottle gourd is the important melon vegetables in summer of China, liked by the people.Scientific research institutions, enterprise that China is engaged in bottle gourd breeding, seed production and operation are numerous, whenever have every year new bottle gourd new variety to breed release.The bottle gourd Varieties of using in the current production is numerous; genetic similarity between commercial variety is more and more high; different name of the same race, xenogenesis phenomenon of the same name are day by day serious; seed intellecture property and quality dispute happen occasionally; be necessary to research and develop a kind of energy early stage, annual, fast, the method for the precise Identification bottle gourd kind true and false and purity; serving the needs of bottle gourd breeding, seed operation and production, and conscientiously protect breeder's legitimum.
China's vegetable variety comprises that the bottle gourd seed true and false and Purity rely on the field phenotype to identify mostly at present.But this evaluation required time is grown (generally needing 50-60 days time), and is subjected to the restriction in season, and the phenotype of crop easily changes with the variation of envrionment conditions, particularly for identifying between the less kind of some morphological differencess that difficulty is larger.
The dna molecular marker technology has been used to differentiate the difference on molecular level between the different varieties at home and abroad.Owing between each kind DNA of same species, having a large amount of polymorphism marks, each kind all has the unique tag that is different from other kind, be the DNA genetic fingerprint that the combination of some DNA fragment specific just is called this kind, the fingerprint fragment of each kind uniqueness consists of the dna fingerprinting of these species.Compare with traditional identifying according to phenotypic character, based on the fingerprint pattern technology of dna level difference, have and to carry out in any stage of plant strain growth, identify the advantages such as impact that are subjected to fast and not genetic expression and envrionment conditions.
The SSR molecule marker is to utilize a large amount of little satellite repetitive sequence design primer that exists in the eukaryotic gene group, by the tumor-necrosis factor glycoproteins of pcr amplification series connection, according to the difference of multiplicity between same species different genotype of little satellite, discloses length polymorphism.SSR is because wide, the stability and polymorphic rate is high, simple to operate, good reproducibility, lower etc. to the DNA specification of quality of distributing is described as the star of s-generation molecule marker.The SSR molecular marking technique has overcome RAPD stability and poor repeatability, AFLP technical fee costliness, complicated operation, to the shortcoming such as the specification of quality of the purity of DNA and restriction endonuclease is very high.
Can obtain species dna sequence dna information and then design and develop the SSR primer by the wholly or in part order-checking to the species gene group.Invention is based on the seed purity method for quick of SSR molecular marking technique, will be to standard bottle gourd seed industry, and protection bottle gourd breeder's legitimate rights and interests and peasant's vital interests promote agricultural produce, increasing peasant income significant.
Summary of the invention
The present invention seeks to, for present bottle gourd variety seeds Purity rely on mostly that phenotype observational technique existing qualification time in field is long, the phenotype that is subjected to seasonal restriction and crop is easily with defectives such as changes in environmental conditions, provide a kind of energy early stage, annual, fast, the detection method of the precise Identification bottle gourd kind true and false and purity; Another object of the present invention provides a kind of test kit of convenient enforcement aforesaid method.
The object of the invention is achieved by the following technical programs.
1, the method for rapid detection bottle gourd variety seeds purity, the method is carried out according to the following steps:
(1) order-checking of the part of bottle gourd genomic dna and the design of SSR primer development:
1) take the main bottle gourd kind " the long melon in Hangzhou " of planting as material, uses sequenator carries out 1/4 flux to its genomic dna once sequencing, obtain 150,253 sequencing sequences;
2) operation Newbler sequence assembly program is carried out sequence assembly to institute's calling sequence and is repeated to remove, and obtains 86,561 of break-even dna sequence dnas;
3) move Mreps 2.5SSR Sequence Identification program, contain the sequence section of SSR on the identification of dna sequence, design simultaneously the PCR primer of crossing over the SSR site; Transfer to the synthetic 100 pairs of SSR primers of Shanghai Sani biotech company;
(2) SSR primer polymorphism information content PIC calculates and the screening of diagnostic SSR combination of primers:
1) extract main bottle gourd kind DNA in 44 current productions, with the DNA of 100 pairs of synthetic these materials of primers difference pcr amplification, the PCR reaction volume is 12.5 microlitres, and annealing temperature is 52 ℃;
2) the amplification situation of every pair of primer of statistics is carried out 0 or 1 assignment to amplified band, and the polymorphism information content that calculates each primer is PIC;
3) carry out comprehensive evaluation from the stability of amplified band, easy resolution degree and three aspects of primer polymorphism PIC index of polymorphic bands, choose LSR011, LSR015, LSR040, LSR045, LSR047, LSR056, LSR063, LSR077 totally 8 pairs of primers as the diagnostic combination of primers, specifically:
(3) preparation of 5 main breed standard DNA of bottle gourd finger printing:
With the listed 8 pairs of diagnostic SSR primers of form increase respectively ' Zhejiang Pu No. 2 ', ' Zhejiang Pu No. 6 ', ' the long melon in Anji ', ' ground, Xiaoshan Pu ' and ' behind the DNA of long melon ' 5, a Hangzhou main breed standard, with amplified production at acrylamide: 8% non-denaturing polyacrylamide gel of methylene diacrylamide=29: 1 carries out electrophoretic separation, silver dyes takes a picture and the record result, obtains the main breed standard finger-print after the Photoshop software processes;
(4) detection of bottle gourd sample to be measured:
Require to differentiate that such as testing sample target variety is one of (three) 5 main breeds of step, then only need with this testing sample carry out extracting genome DNA, and carry out same step (two) 1 with the listed 8 pairs of SSR primers of above table) PCR react, gel electrophoresis, the silver of step (three) dye, obtain finger printing after, compare with 5 main breed standard finger-prints, with the true and false of determining testing sample and and then calculate its purity; As require to differentiate that target variety is non-above-mentioned 5 main breeds, then need testing sample and the standard model that requires to differentiate target variety, prepare respectively finger printing and compare, with the true and false of determining the testing sample seed and and then calculate as follows its seed purity:
Seed purity=(detecting gained pure dan number/detection seed sum) * 100%.
2, the test kit of rapid detection bottle gourd variety seeds purity, this test kit comprise box body and 12 eppondorf pipe, 25mM MgCl are housed respectively in 4 therein
210mM dNTP, 10X PCR damping fluid and 5U/ μ L Taq archaeal dna polymerase is characterized in that in all the other 8 eppondorf pipes, be equipped with respectively and contain the diagnostic SSR detection primer LSR011 that forward and reverse concentration is 1mM, LSR015, LSR040, LSR045, LSR047, LSR056, LSR063, LSR077; In addition, also be equipped with in the box body and be printed on the listed bottle gourd of claim 1 step (three) ' Zhejiang Pu No. 2 ', ' Zhejiang Pu No. 6 ', ' G5-3 ', ' G26 ' and ' picture of long melon ' 5, a Hangzhou main breed standard DNA finger printing.
The invention has the beneficial effects as follows:
(1) 8 pairs of diagnostic SSR combination of primers filtering out of application autonomous design have strengthened the ability of differentiating approximate kind.The approximate kind of process increases to compare (Fig. 1) and detect in a large number and facts have proved that this overlaps primer and can effectively distinguish the upper common different bottle gourd genotype of production, can satisfy the needs that main bottle gourd kind in the current production carried out true and false detection.
(2) by the Direct Identification to the bottle gourd genetic material, greatly improved the accuracy of identifying.The present invention makes the evaluation of bottle gourd seed purity carry out at dna level, avoided the indirectly error that may bring of authentication method such as phenotypic evaluation, biochemical identification, has easy to operately, and the characteristics of good reproducibility have reliability and authority highly.
(3) efficient and the standardization level that detect have been improved.The present invention will detect with primer and detection reagent and provide so that kit form is supporting, and detected result is reliable and stable, is easy to realize stdn, and only need 4-5 hour detection time, once can detect simultaneously duplicate samples up to a hundred.General seed quality inspection chamber only need possess conventional molecular biology equipment just can carry out actual detection by the schedule of operation that provides.PCR reaction component (MgCl
2, Buffer, dNTP, Taq enzyme) both can be provided by this test kit, also can be purchased separately by user oneself, easy to use, and testing cost is cheap, is about the sixth (seeing embodiment 3) of traditional phenotypic evaluation.
Description of drawings
Fig. 1 is the standard DNA finger printing of 5 main breeds of bottle gourd.
1:LSR011 wherein, 2:LSR015,3:LSR040,4:LSR045,5:LSR047,6:LSR056,7:LSR063,8:LSR077; Arrow shows the Main Differences part of each kind dna fingerprint.
Embodiment
The present invention also is described in further detail by reference to the accompanying drawings by following examples, but should be appreciated that the present invention is not placed restrictions on by following content.
Sequenator: German Roche Holding Ag ' 454 ' sequenator.
Embodiment 1:(uses test kit to requiring to differentiate that target variety is the detection of one of main breed testing sample)
Carry out according to the following steps:
(1) DNA extraction: seed to be checked is disseminated in vermiculite is housed: in the seedling culture hole plate of peat=1: 2 matrix, place 25-30 ℃ to grow seedlings, when treating that first pair of true leaf is open and flat, get blade 0.1 gram by individual plant, add the liquid nitrogen grind into powder, extract DNA with conventional CTAB method, for subsequent use;
(2) SSR primer amplification and electrophoresis detection: 8 pairs of SSR diagnostic primers that provide with test kit carry out pcr analysis, and reaction volume is 12.5 μ L; Each component of PCR is 10 * buffer, 1.25 μ L, 25mM MgCl
20.75 μ L, 2.5mM dNTPs 1 μ L, Taq enzyme (5U/ μ L) 0.1 μ L, template DNA 10ng adds water to 12.5 μ L; The SSR response procedures is: 94 ℃ of denaturation 3min, and 94 ℃ of sex change 30sec, 52 ℃ of annealing 30sec, 72 ℃ are extended 50sec, circulate 35 times, and last 72 ℃ are extended 5min; In the enterprising performing PCR amplification of PTC-225 amplification instrument, amplified production is at acrylamide: 8% non-denaturing polyacrylamide gel of methylene diacrylamide=29: 1 carries out electrophoretic separation, then carry out silver and dye colour developing, digital camera photographic recording result obtains the dna fingerprinting of material to be checked;
(3) dna fingerprinting comparison and seed purity are calculated: the bottle gourd ' Zhejiang Pu No. 2 ' that dna fingerprinting and the test kit of material to be checked provided, ' Zhejiang Pu No. 6 ', ' the long melon in Anji ', ' Xiaoshan Pu ' reach that ' long melon ' 5, a Hangzhou main breed standard DNA finger printing is compared; The target variety that for example provides material person to be checked to require to differentiate is ' the long melon in Hangzhou ' of one of 5 main breeds, and the finger printing comparison result both match fully, then this material to be checked is real ' the long melon in Hangzhou ', and and then use formula: seed purity=(detecting gained pure dan number/detections seed total) * 100% calculates the purity of seed to be checked; Otherwise be pseudo-seed.
Embodiment 2:(uses test kit to requiring to differentiate that target variety is the detection of one of non-main breed testing sample)
Carry out according to the following steps:
(1) DNA extraction: with the standard seed of seed to be detected and requirement discriminating target variety, disseminate respectively in vermiculite is housed: in the seedling culture hole plate of peat=1: 2 matrix, place 25-30 ℃ to grow seedlings, when treating that first pair of true leaf is open and flat, get blade 0.1 gram by individual plant, add the liquid nitrogen grind into powder, extract DNA with the CTAB method, for subsequent use;
(2) SSR primer amplification and electrophoresis detection: with embodiment 1;
(3) respectively according to seed to be detected and standard variety seed pcr amplification and electrophoresis detection result, be built into the dna fingerprinting of seed to be detected and standard variety seed;
(4) dna fingerprinting comparison and seed purity are calculated: both dna fingerprintings are compared, the identical pure dan that is of the two banding pattern, and and then use formula: seed purity=(, detection gained pure dan number/detection seed sum) * 100%, calculate the purity of seed to be checked; Otherwise be pseudo-seed.
Embodiment 3:(uses the simultaneous test of tradition and the inventive method detection bottle gourd seed purity)
Require to differentiate that target variety is the one of 5 main breeds, then undertaken by embodiment 1 method;
Require to differentiate that target variety is the one of non-main breed, then undertaken by embodiment 2 methods;
Traditional detection method carries out according to the following steps:
1, get respectively kind to be detected and require to differentiate that the standard seed of target variety is seeded in vermiculite: in the 8X8cm nutrition pot of peat=1: 2 matrix, each nutrition pot is broadcast 2 seeds, treats to carry out thinning when cotyledon is open and flat, and each nutrition pot stays 1 strain; Be colonizated in the booth in 1 heart stage of 2 leaves, line-spacing 70cm, spacing in the rows 40cm is more than every kind 100 strains;
2, cultivation technique is carried out daily field management routinely, and begins to carry out the characters of seedling stage contrast;
3, enter the feature that the after date of bearing fruit examines this standard variety, and the difference of variety characteristic more to be measured and standard variety one by one, to determine the true and false of seed, use following formula after detection is all finished: seed purity=(detecting gained pure dan number/detection seed sum) * 100% calculates the seed purity of each kind to be measured.
The comparing result of table 1 the present invention and traditional technique in measuring bottle gourd seed purity
Claims (2)
1. the method for rapid detection bottle gourd variety seeds purity is characterized in that carrying out according to the following steps:
(1) order-checking of the part of bottle gourd genomic dna and the design of SSR primer development:
1) take the main bottle gourd kind " the long melon in Hangzhou " of planting as material, uses sequenator carries out 1/4 flux to its genomic dna once sequencing, obtain 150,253 sequencing sequences;
2) operation Newbler sequence assembly program is carried out sequence assembly to institute's calling sequence and is repeated to remove, and obtains 86,561 of break-even dna sequence dnas;
3) move Mreps 2.5SSR Sequence Identification program, contain the sequence section of SSR on the identification of dna sequence, design simultaneously the PCR primer of crossing over the SSR site; Transfer to the synthetic 100 pairs of SSR primers of Shanghai Sani biotech company;
(2) SSR primer polymorphism information content PIC calculates and the screening of diagnostic SSR combination of primers:
1) extract main bottle gourd kind DNA in 44 current productions, with the DNA of 100 pairs of synthetic these materials of primers difference pcr amplification, the PCR reaction volume is 12.5 microlitres, and annealing temperature is 52 ℃;
2) the amplification situation of every pair of primer of statistics is carried out 0 or 1 assignment to amplified band, and the polymorphism information content that calculates each primer is PIC;
3) carry out comprehensive evaluation from the stability of amplified band, easy resolution degree and three aspects of primer polymorphism PIC index of polymorphic bands, choose LSR011, LSR015, LSR040, LSR045, LSR047, LSR056, LSR063, LSR077 totally 8 pairs of primers as the diagnostic combination of primers, specifically:
(3) preparation of 5 main breed standard DNA of bottle gourd finger printing:
With the listed 8 pairs of diagnostic SSR primers of form increase respectively ' Zhejiang Pu No. 2 ', ' Zhejiang Pu No. 6 ', ' the long melon in Anji ', ' ground, Xiaoshan Pu ' and ' behind the DNA of long melon ' 5, a Hangzhou main breed standard, with amplified production at acrylamide: 8% non-denaturing polyacrylamide gel of methylene diacrylamide=29: 1 carries out electrophoretic separation, silver dyes takes a picture and the record result, obtains the main breed standard finger-print after the Photoshop software processes;
(4) detection of bottle gourd sample to be measured:
Require to differentiate that such as testing sample target variety is one of (three) 5 main breeds of step, then only need with this testing sample carry out extracting genome DNA, and after the PCR reaction, gel electrophoresis, the silver that carry out same step (two) (1) with the listed 8 pairs of SSR primers of above table dyes, obtains finger printing, compare with 5 main breed standard finger-prints, with the true and false of determining testing sample and and then calculate its purity; As require to differentiate that target variety is non-above-mentioned 5 main breeds, then need testing sample and the standard model that requires to differentiate target variety, prepare respectively finger printing and compare, with the true and false of determining the testing sample seed and and then calculate as follows its seed purity:
Seed purity=(detecting gained pure dan number/detection seed sum) * 100%.
2. the test kit of rapid detection bottle gourd variety seeds purity, this test kit comprise box body and 12 eppendorf pipes, 25mM MgCl are housed respectively in 4 therein
210mM dNTP, 10X PCR damping fluid and 5U/ μ L Taq archaeal dna polymerase is characterized in that in all the other 8 eppendorf pipes, be equipped with respectively and contain the diagnostic SSR detection primer LSR011 that forward and reverse concentration is 1mM, LSR015, LSR040, LSR045, LSR047, LSR056, LSR063, LSR077; In addition, also be equipped with in the box body and be printed on the listed bottle gourd of claim 1 step (three) ' Zhejiang Pu No. 2 ', ' Zhejiang Pu No. 6 ', ' the long melon in Anji ', ' ground, Xiaoshan Pu ' and ' picture of long melon ' 5, a Hangzhou main breed standard DNA finger printing;
Described diagnostic SSR detects primer:
。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110207456 CN102251042B (en) | 2011-07-22 | 2011-07-22 | Method for quickly detecting purity of seeds of bottle gourd varieties and kit used by same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110207456 CN102251042B (en) | 2011-07-22 | 2011-07-22 | Method for quickly detecting purity of seeds of bottle gourd varieties and kit used by same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102251042A CN102251042A (en) | 2011-11-23 |
| CN102251042B true CN102251042B (en) | 2013-02-06 |
Family
ID=44978610
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110207456 Expired - Fee Related CN102251042B (en) | 2011-07-22 | 2011-07-22 | Method for quickly detecting purity of seeds of bottle gourd varieties and kit used by same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102251042B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667487B (en) * | 2013-12-03 | 2015-10-21 | 浙江省农业科学院 | Based on the method for EST-SSR Marker Identification Loquat Cultivars |
| CN110923357B (en) * | 2019-12-31 | 2022-04-19 | 浙江省农业科学院 | Bottle gourd core molecular marker set developed based on KASP technology and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701249A (en) * | 2009-11-19 | 2010-05-05 | 浙江省农业科学院 | Molecular marker method for detecting resistance of powdery mildew of bottlegourd and kit thereof |
-
2011
- 2011-07-22 CN CN 201110207456 patent/CN102251042B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701249A (en) * | 2009-11-19 | 2010-05-05 | 浙江省农业科学院 | Molecular marker method for detecting resistance of powdery mildew of bottlegourd and kit thereof |
Non-Patent Citations (3)
| Title |
|---|
| 《AFLP分子标记技术在浙蒲2号种子纯度快速鉴定中的应用》;王玲平等;《浙江农业学报》;20080325;第20卷;第84－87页 * |
| 刘成平.《瓠瓜EST-SSR、SRAP、ISSR和RAPD反应体系的建立及其纯度鉴定应用的研究》.《中国优秀硕士学位论文全文数据库农业科技辑(月刊)》.2009,(第02期),第7-8页. * |
| 王玲平等.《AFLP分子标记技术在浙蒲2号种子纯度快速鉴定中的应用》.《浙江农业学报》.2008,第20卷第84－87页. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102251042A (en) | 2011-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101684487B (en) | Method for identifying industrially cultivated strains of hypsizygus marmoreus by using SSR molecular marker | |
| CN102321768A (en) | Method for identifying camellia oleifera cultivar and special primer and kit thereof | |
| Jedrzejczyk | Study on genetic diversity between Origanum L. species based on genome size and ISSR markers | |
| CN108486275B (en) | Method for identifying begonia varieties by SSR (simple sequence repeat) fingerprint spectrum and application of method | |
| CN103194539A (en) | Method for identifying ramie variety by simple sequence repeat (SSR) molecular marker | |
| CN102251042B (en) | Method for quickly detecting purity of seeds of bottle gourd varieties and kit used by same | |
| CN101671730B (en) | Method for rapidly detecting seed purity of asparagus bean cultivars and reagent kit thereof | |
| CN105861729A (en) | Molecular marker combination for Litopenaeus vannamei germplasm identification and application thereof | |
| CN112725521B (en) | Dendrobium chrysotoxum SSR molecular marker primer composition and application thereof | |
| KR101423299B1 (en) | A method for identifying melon varieties using microsatellites markers | |
| CN104975083A (en) | Primers for rapid identification of two bemisia tabaci cryptic species of MEAM1 and MED and identification method thereof | |
| CN104372096B (en) | A kind of Corchorus olitorius L. microsatellite DNA mark finger printing and application thereof | |
| Li et al. | Establishment of molecular identity cards for Cucumis melo cultivars using ssr markers | |
| CN102296124B (en) | A kind of RAPD of utilization distinguishes the method for jujube kind fast | |
| CN107012253B (en) | Method for identifying female parent of cultivated oat | |
| KR101359542B1 (en) | Microsatellite primer sets for discriminating cultivars of peach and uses thereof | |
| KR101301863B1 (en) | A method for identifying lettuce varieties using microsatellite primer set | |
| CN106755288B (en) | Molecular identification method of lotus leaf of tongshan | |
| CN105112542B (en) | A kind of method identified with SRAP molecular labelings rescuebrome kind | |
| CN107130034A (en) | A kind of method that utilization SSR marker identifies flat Europe hybrid hazel kind | |
| CN112176088B (en) | SSR primer group for distinguishing litchi varieties and application thereof | |
| CN102181551A (en) | Carmine spider mite SCAR (sequence characterized amplified region) mark and specificity PCR (polymerase chain reaction) detection method thereof | |
| CN113481313B (en) | Multiple fluorescence SSR (simple sequence repeat) labeled primers and method for identifying three Chinese torreya varieties | |
| CN116121438B (en) | Molecular marker primer group and kit for identifying mulberry new variety 'Yuesang 162' for silkworm raising leaves and application of molecular marker primer group and kit | |
| CN113981124B (en) | Sakura SSR molecular marker primer and application thereof in identification of 42 sakura varieties |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130206 Termination date: 20210722 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |