CN101914629A - Erigeron breviscapus DNA polymorphism detection method and application thereof - Google Patents
Erigeron breviscapus DNA polymorphism detection method and application thereof Download PDFInfo
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Abstract
The invention provides an erigeron breviscapus DNA polymorphism detection method and application thereof. In the invention, an amplified fragment length polymorphism technology is used for erigeron breviscapus DNA polymorphism detection, an optimum primer combination is screened out, and reaction condition is optimized. The method can be effectively applied to the inheritance breeding, the resource protection, the introduction domestication, the breed optimization and the like of erigeron breviscapus.
Description
Technical field
The present invention relates to dna polymorphism detection method of Erigeron breviscapus (Vant.) Hand.-Mazz. and uses thereof, particularly, the dna polymorphism that the present invention adopts the amplified fragment length polymorphism technology to carry out Erigeron breviscapus (Vant.) Hand.-Mazz. detects
Background technology
Amplified fragment length polymorphism (Amplified fragment length polymorphism, AFLP) be in random amplified polymorphism (RAPD) and the technical dna polymorphism detection technique that grows up of restriction fragment length polymorphism (RFLP), have RFLP technology high duplication and the simple and efficient characteristics of RAPD technology, need must not prepare probe as rflp analysis, and the same detection to genome polymorphism with the RAPD mark does not need to know its genomic sequence signature, has remedied the defective of RAPD technology poor repeatability simultaneously.Compare with other labeling techniques based on PCR, the AFLP technology can detect a large amount of sites and polymorphism mark simultaneously.This technology has been successfully used to genetic diversity research, and the research of germ plasm resource evaluation aspect makes up genetic map and finger printing etc.AFLP can more fully disclose hereditary difference than RAPD, ISSR, and finger printing is more detailed.
Erigeron breviscapus (Vant.) Hand.-Mazz. [Erigeron breviscapus (Vant.) Hand.-Mazz], popular name be Herba Erigerontis, Herba Erigerontis, oil lamp grass, lamp top grass, be exposed to the sun, eastern chrysanthemum, two sunflowers etc., originate in the Hunan, Guangxi, Guizhou, Yunnan, provinces and regions such as Tibet, the Zhong Shan and the inferior high mountain that are common in height above sea level 1200-3500 rice are opened spacious hillside, the meadow border, be herbal medicine commonly used among the people, all herbal medicine, the tool expelling cold and relieving exterior syndrome, dispel rheumatism, the effect of activating collaterals to relieve pain.
Up to now, mainly concentrate on the extraction and the evaluation of chemical ingredients at the research of Erigeron breviscapus (Vant.) Hand.-Mazz., physiology and appearance, cultivation technique and isozyme aspect, and less for the research work of its molecular level.In view of Erigeron breviscapus (Vant.) Hand.-Mazz. has important medical value; can be used as important economic plants; therefore; obtain the Erigeron breviscapus (Vant.) Hand.-Mazz. genetic data of high information quantity; grope to set up its dna molecular finger printing, all be significant at aspects such as the conservation of resources of Erigeron breviscapus (Vant.) Hand.-Mazz., introduction and acclimatization, genetic breeding, new variety protections.
Summary of the invention
Therefore, the dna polymorphism detection method that the purpose of this invention is to provide a kind of Erigeron breviscapus (Vant.) Hand.-Mazz..
Another object of the present invention provides the purposes of aforesaid method.
The objective of the invention is to realize by the following technical solutions.
The invention provides a kind of dna polymorphism detection method of Erigeron breviscapus (Vant.) Hand.-Mazz., described method adopts the amplified fragment length polymorphism technology that the dna polymorphism of Erigeron breviscapus (Vant.) Hand.-Mazz. is detected, and specifically may further comprise the steps: 1) enzymolysis of Erigeron breviscapus (Vant.) Hand.-Mazz. genomic dna be connected; 2) preselected amplification; 3) select amplification; Wherein, the primer sequence of selecting in the step 3) to increase comprises:
EcoRI be 5 '-GACTGCGTACCAATTCAGG-3 ' or 5 '-GACTCCGTACCAATTCCGA-3 ' and
Mse I is 5 '-GATGAGTCCTGAGTAACAA-3 '.
Preferably, described method steps 1) enzymolysis in is that the Erigeron breviscapus (Vant.) Hand.-Mazz. genomic dna is carried out the EcoR1/Mse1 double digestion, and it is as follows that described enzyme is cut system: EcoR1 enzymic digestion system (20ul system/sample) is EcoR1 1ul (10U/ul), 10 * damping fluid 2ul, sample (total DNA) 5ul, ddH
2O12ul; Mse1 enzymic digestion system (20ul system/sample) is Mse1 2ul (1U/ul), 10 * damping fluid 2ul, and the EcoR1 enzyme is cut product 15ul, ddH
2O 1ul; The endonuclease reaction condition is as follows: EcoR1 enzymic digestion condition be 37 ℃ 2 hours, 65 ℃ 30 minutes, termination reaction; Mse1 enzymic digestion condition: 65 ℃ 2 hours, 80 ℃ 30 minutes, termination reaction.
Preferably, adopt described method steps 1) and cut segmental end with the lower sub ligase enzyme:
The EcoRI joint is 5 '-CTCGTAGACTGCGTACC-3 ' (forward),
3 '-CATCTGACGCATGGTTAA-5 ' (oppositely);
The MseI joint is 5 '-GACGATGAGTCCTGAG-3 ' (forward),
3 '-TACTCAGGACTCAT-5 ' (oppositely);
The ligation condition is as follows: the T4DNA ligase enzyme, 22 ℃ 3 hours, 65 ℃ 10 minutes, termination reaction.
Preferably, the connection product that obtains described method steps 1) carry out step 2 as dna profiling after through 20 times of dilutions) in preselected amplification.
Preferably, the primer sequence that carries out preselected amplification described method steps 2) comprises:
EcoRI:5 '-GTAGACTGCGTACCAATTC-3 ' and
Mse?I：5’-GACGATGAGTCCTGAG-3’。
Preferably, described method steps 2) amplification system (20ul system/sample) of preselected amplification is template DNA 4ul in, primer 1ul, AFLP Core Mix 15ul, ddH
2O 13.8ul, MgCl
21.6ul, dNTPs (2.5Mm) 1.6ul, Taq enzyme (1U/ul) 1ul, 10 * damping fluid 2ul; The PCR reaction conditions is as follows: 94 ℃ of 2min, 94 ℃ of 20s, 56 ℃ of 30s, totally 20 circulations, 72 ℃ of 2min, 60 ℃ of 30min.
Preferably, the product that preselected amplification obtains described method steps 2) carries out selection amplification in the step 3) as dna profiling after through 20 times of dilutions.
Preferably, described method steps 3) selecting the amplification system (20ul system/sample) of amplification in is dna profiling 4ul, primer 1ul, AFLP Core Mix 15ul; The PCR reaction conditions is as follows: 94 ℃ of 2min, 94 ℃ of 20s, 66 ℃ of 30s, 1 ℃ of every cycle down, totally 10 circulations, 72 ℃ of 2min, 94 ℃ of 20s, 56 ℃ of 30s, totally 20 circulations, 72 ℃ of 2min, 60 ℃ of 30min.
Preferably, described method also comprises the step that adopts sex change electrophoresis and silver staining method to detect resulting amplified production in the step 3).
In addition, the invention provides the application of aforesaid method in setting up Erigeron breviscapus (Vant.) Hand.-Mazz. dna molecular finger printing.
The present invention also provides the application of aforesaid method in genetic breeding, conservation of resources, introduction and acclimatization or the optimized varieties of Erigeron breviscapus (Vant.) Hand.-Mazz..
This shows, the invention provides a kind of dna polymorphism detection method of Erigeron breviscapus (Vant.) Hand.-Mazz., the dna polymorphism that adopts amplified fragment length polymorphism (AFLP) technology to carry out Erigeron breviscapus (Vant.) Hand.-Mazz. detects, according to band number and the distribution situation in 100～500bp scope, and polymorphic bands number, filter out only combination of primers, can obtain the Herba Erigerontis genetic data of high information quantity.In addition, the present invention has also optimized the various reaction conditionss that carry out the AFLP reaction.By method provided by the invention; can obtain the Herba Erigerontis genetic data of high information quantity; set up its dna molecular finger printing; genetic breeding, conservation of resources, introduction and acclimatization, optimized varieties at Herba Erigerontis play a key effect, to promoting Herba Erigerontis plantation industrialization, standardization and scientific being significant.
Description of drawings
Describe embodiment of the present invention in detail below in conjunction with accompanying drawing, wherein:
Fig. 1 by among the embodiment 1 the genome of the total DNA of extraction partly detect electrophoresis result figure, wherein turning left from the right side is followed successively by Marker, sample 1-7.
Fig. 2 cuts the part electrophoresis result figure of genomic dna for adopting the Ecor1 enzyme among the embodiment 1, wherein turns left from the right side and is followed successively by Marker, sample 1-20.
Fig. 3 carries out the part amplification electrophoresis result figure that the AFLP reaction obtains for adopting primer E3M4 among the embodiment 2.
Fig. 4 carries out the part amplification electrophoresis result partial enlarged drawing that the AFLP reaction obtains, wherein band specific amplified in sample 1 of arrow 1 indication for adopting primer E1M5 among the embodiment 2; The band of arrow 2 indications increases in sample 1,5,15,17; The band of arrow 3 indications increases in sample 1,17; Band specific amplified in sample 2 of arrow 4 indications; The band of arrow 5 indications is not amplification in sample 1; The band of arrow 6 indications increases in sample 1,15,17; The band of arrow 7 is not amplification in sample 1; Band specific amplified in sample 21 of arrow 8 indications.
Fig. 5 is to the AFLP cluster analysis result figure of 40 parts of laboratory samples among the embodiment 2.
Embodiment
Followingly the present invention is described with reference to specific embodiment.It will be appreciated by those skilled in the art that these embodiment only are used to illustrate the present invention, the scope that it does not limit the present invention in any way.
Embodiment 1Total DNA extraction
Present embodiment has extracted total DNA of 40 parts of Herba Erigerontis experiment materials respectively, to be ready for use on AFLP these materials is carried out genetic diversity and molecular marking fingerprint analysis and research.
Table 1 experiment material title and source
Concrete DNA extraction method is as follows: the young leaflet tablet of getting the 0.2g experiment material adds liquid nitrogen and is ground to Powderedly, adds 1ml and extracts damping fluid (containing 2%CTAB) and place 65 ℃ of insulation 1h, constantly shakes up therebetween.Take out the back centrifugal 10min of 10000rpm (4 ℃).After the centrifugal end supernatant liquor is forwarded in another clean centrifuge tube, add isopyknic chloroform: primary isoamyl alcohol (24: 1) constantly shakes up therebetween.Leave standstill moments later the centrifugal 10min of 10000rpm (4 ℃), the taking-up supernatant liquor adds isopyknic Virahol and places-20 ℃ to precipitate about 30min.The centrifugal 5min of 5000rpm (4 ℃), abandoning supernatant, the alcohol washing precipitation of adding 1ml 75% repeats secondary.Then that centrifuge tube is aseptic air-dry to transparence in room temperature, add 30 μ l TE or ddH
2The O dissolution precipitation.Add after 2 μ l RNA enzymes place 37 ℃ of incubator 1-2h with centrifuge tube ,-20 ℃ of preservations, last sample 3 μ l, 0.8% agarose gel electrophoresis detects DNA concentration and quality.
Conjugated double bond is all arranged in the purine of nucleic acid, the pyrimidine, there is intensive to absorb to UV-light, about 1.80, low then illustrate that protein may not eliminate in the preparation, height then illustrates to also have RNA in the preparation to natural double-stranded DNA at the absorption value at 260nm place and 280nm place absorption value ratio (A260/A280).Total DNA sample major part that the present invention adopts the CTAB method to extract all presents oyster white, and a few has color, may be that aldehydes matter is not removed totally, and browning appears in material, or pigment is not removed totally.With (the reference " molecular cloning experiment guide (third edition) " of 0.8% agarose gel electrophoresis, Huang Peitang etc. translate, and Science Press in September, 2002 publishes) detect the results are shown in Figure 1, DNA is the band of a complete display, not significantly hangover, RNA is with to have small part not degrade completely.Generally, the dna fragmentation size is consistent, (carries out double digestion with EcoRI and MseI, EcoRI enzymic digestion condition: 65 ℃ of 30min termination reactions of 37 ℃ of 2hrs through calculating and endonuclease reaction detection; MseI enzymic digestion condition: 80 ℃ of 30min termination reactions of 65 ℃ of 2hrs) (the results are shown in Figure 2) shows that also content (100ng/ μ l) and the quality of DNA have also reached the AFLP experimental requirements.
Embodiment 2The AFLP reaction
Present embodiment specifically may further comprise the steps for the genomic dna that extracts among the embodiment 1 being carried out the AFLP reaction:
1) enzyme is cut and is connected:
The genomic dna that extracts among the embodiment 1 is carried out the EcoR1/Mse1 double digestion, and enzyme cuts system and condition is as follows:
(1) EcoRI enzymic digestion system (20ul system/sample): EcoRI 1ul (10U/ul), 10 * damping fluid 2ul, sample (total DNA) 5ul, ddH
2O 12ul;
EcoR1 enzymic digestion condition: 37 ℃ of 2h, 65 ℃ of 30min, termination reaction;
(2) MseI enzymic digestion system (20ul system/sample): MseI 2ul (1U/ul), 10 * damping fluid 2ul, sample (the EcoRI enzyme is cut product) 15ul, ddH
2O 1ul;
Mse1 enzymic digestion condition: 65 ℃ of 2 hours h, 80 ℃ of 30 minutes min, termination reaction;
Enzyme is cut product be connected with the synthetic linker fragment, joint sequence wherein is as follows:
(1) EcoRI joint: 5 '-CTCGTAGACTGCGTACC-3 ' (forward),
3 '-CATCTGACGCATGGTTAA-5 ' (oppositely);
(2) MseI joint: 5 '-GACGATGAGTCCTGAG-3 ' (forward),
3 '-TACTCAGGACTCAT-5 ' (oppositely).
Condition of contact is: T4DNA ligase enzyme, 22 ℃ of 3h, 65 ℃ of 10min, termination reaction.
2) preselected amplification
After 5ul connected mixed solution and isopyknic limited enzymatic hydrolysis liquid (EcoR1 and Mse1) mixes, 16 ℃ of following incubated overnight, get the 5ul mixed solution, add 10mmol/L Tris (pH 8.5) then, by the template DNA of 20 times of these mixed solutions of dilution as preselected amplification to 100ul.
Pre-amplification primer is: EcoRI:5 '-GTAGACTGCGTACCAATTC-3 ' and
Mse?I：5’-GACGATGAGTCCTGAG-3’。
Carry out preselected amplification system (20ul system/sample): template DNA 4ul, primer (Mse1/EcoR1) 1ul, AFLP Core Mix 15ul, ddH
2O 13.8ul, MgCl
21.6ul, dNTPs (2.5Mm) 1.6ul, Taq enzyme (1U/ul) 1ul, 10 * damping fluid 2ul.
Preselected amplification reaction condition: 94 ℃ of 2min, 94 ℃ of 20s, 56 ℃ of 30s, totally 20 circulations, 72 ℃ of 2min, 60 ℃ of 30min.
3) select amplification
The template DNA that increases as selection after the product that preselected amplification obtains dilutes 20 times.
Primer sequence is respectively 7 pairs of combination of primers listing in the table 2.
Select amplification system (20ul system/sample): template DNA 4ul, primer (Mse1-XXX/EcoR1-XXX) 1ul, AFLP Core Mix 15ul;
Select amplification reaction condition: 94 ℃ of 2min, 94 ℃ of 20s, 66 ℃ of 30s, 1 ℃ of every cycle down, totally 10 circulations, 72 ℃ of 2min, 94 ℃ of 20s, 56 ℃ of 30s, totally 20 circulations, 72 ℃ of 2min, 60 ℃ of 30min.
4) detect amplified production
Show band by sex change electrophoresis and silver staining method.
By denaturing polyacrylamide gel electrophoresis the amplified reaction product is detected: sample solution 20ul, marker (Size standard-600) 0.2ul, sample 3ul (diluting 10 times).
Silver staining method is as follows:
1) 200ml 10% alcohol (ethanol)+0.5% acetate, 3-5min;
2)200ml?0.2％AgNO
3，5-8min；
3) washing 1min;
4) 200ml 0.2%Na
2S
2O
3, 1min (adds 1%Na in 200ml water
2S
2O
3400ul);
5) 200ml 1.5%NaOH+1% formaldehyde colour developing;
6) washing.
Choosing the band in the 100bp-500bp scope, is that 0 method writes down electrophoretic band according to having amplified band to be designated as 1 on the identical migration position, not having band, only clear, repeatably amplified band just is recorded.The AFLP data matrix that obtains is further analyzed with NTSYSpc-2.02 software.The primer result of different choice amplification is as shown in table 2.
Table 2 AFLP primer screening and amplification
Each primer sequence in the table 2 is as follows:
E1(SEQ.ID.NO：1)：5’-GACTGCGTACCAATTCAGG-3’；
E2(SEQ.ID.NO：2)：5’-GACTGCGTACCAATTCACT-3’；
E3(SEQ.ID.NO：3)：5’-GACTCCGTACCAATTCAGA-3’；
E4(SEQ.ID.NO：4)：5’-GACTCCGTACCAATTCACT-3’；
E5(SEQ.ID.NO：5)：5’-GACTCCGTACCAATTCCGA-3’；
M2(SEQ.ID.NO：6)：5’-GATGAGTCCTGAGTAACTC-3’；
M4(SEQ.ID.NO：7)：5’-GATGAGTCCTGAGTAACGA-3’；
M5(SEQ.ID.NO：8)：5’-GATGAGTCCTGAGTAACAA-3’。
The present invention filters out the AFLP reaction of best combination of primers E1M5 or E5M5 carry out to(for) Herba Erigerontis from numerous known combination of primers.
As can be seen from Table 2,7 pairs of primer coamplifications go out 352 bands, and polymorphic bands is 151, accounts for 42.89% of total band, and amplified band and polymorphic bands that primer E1M5 amplification obtains are maximum, and the pleomorphism site ratio reaches 52.63%.The part amplification of primer E3M4 as shown in Figure 3.Primer E1M5 sees Fig. 4 to the specificity electrophoretic band partial enlarged drawing of sample 1,2,3,15,17,21.
On the whole, can detect with the AFLP method and obtain each sample room good polymorphism is arranged.The AFLP fingerprint characteristic is very obvious, especially the AFLP figure spectrum signature of six new seed selection kind materials (promptly in the table 1 sample No. 1, No. 2, No. 3, No. 15, No. 17, No. 21) has notable difference, and be different from other Herba Erigerontis material, illustrate that thus these material genetic material (gene) are different from other material.The AFLP data matrix that obtains is further analyzed with NTSYSpc-2.02 software, and cluster analysis result is seen Fig. 5.As seen from Figure 5,40 parts of material genetic distances that supply examination are between 0.05-0.26, at genetic distance is that 0.186 (GS=0.814) locates, 40 samples for examination can be divided into 5 cluster groups, wherein differing greatly of No. 19 samples, No. 20 samples, No. 36 samples and other each sample room can respectively be classified it as a class separately; Sample 11 and 14 is classified as a class; All the other 35 samples are classified as a class.From the genetic diversity angle, the diversity index of these materials is not high, show that sibship is nearer between these materials, their some proterties such as output, effectively pharmaceutical ingredient content, ecological suitability difference or variation are only caused by a part of gene variation.
Claims (10)
1. the dna polymorphism detection method of an Erigeron breviscapus (Vant.) Hand.-Mazz. is characterized in that, described method is the amplified fragment length polymorphism technology, may further comprise the steps:
1) enzymolysis of Erigeron breviscapus (Vant.) Hand.-Mazz. genomic dna be connected;
2) preselected amplification;
3) select amplification.
The primer sequence of selecting in the wherein said step 3) to increase comprises:
EcoRI be 5 '-GACTGCGTACCAATTCAGG-3 ' or 5 '-GACTCCGTACCAATTCCGA-3 ' and
Mse I is 5 '-GATGAGTCCTGAGTAACAA-3 '.
2. method according to claim 1 is characterized in that, described method steps 1) in enzymolysis be that the Erigeron breviscapus (Vant.) Hand.-Mazz. genomic dna is carried out the EcoR1/Mse1 double digestion;
Preferably, it is as follows that described enzyme is cut system: EcoR1 enzymic digestion system (20ul system/sample) is EcoR1 1ul (10U/ul), 10 * damping fluid 2ul, sample (total DNA) 5ul, ddH
2O 12ul; Mse1 enzymic digestion system (20ul system/sample) is Mse1 2ul (1U/ul), 10 * damping fluid 2ul, and the EcoR1 enzyme is cut product 15ul, ddH
2O 1ul;
More preferably, described endonuclease reaction condition is as follows: EcoR1 enzymic digestion condition be 37 ℃ 2 hours, 65 ℃ 30 minutes, termination reaction; Mse1 enzymic digestion condition be 65 ℃ 2 hours, 80 ℃ 30 minutes, termination reaction.
3. method according to claim 1 and 2 is characterized in that, adopts and cuts segmental end with the lower sub ligase enzyme:
The EcoRI joint is 5 '-CTCGTAGACTGCGTACC-3 ' (forward),
3 '-CATCTGACGCATGGTTAA-5 ' (oppositely);
The MseI joint is 5 '-GACGATGAGTCCTGAG-3 ' (forward),
3 '-TACTCAGGACTCAT-5 ' (oppositely);
Preferably, described ligation condition is as follows: the T4DNA ligase enzyme, 22 ℃ 3 hours, 65 ℃ 10 minutes, termination reaction.
4. according to each described method in the claim 1 to 3, it is characterized in that described method steps 1) in the connection product that obtains carry out step 2 as dna profiling after through 20 times of dilutions) in preselected amplification.
5. according to each described method in the claim 1 to 4, it is characterized in that described method steps 2) in carry out preselected amplification primer sequence comprise:
EcoRI:5 '-GTAGACTGCGTACCAATTC-3 ' and
Mse?I：5’-GACGATGAGTCCTGAG-3’；
Preferably, described method steps 2) amplification system (20ul system/sample) of preselected amplification is template DNA 4ul in, primer 1ul, AFLP Core Mix 15ul, ddH
2O 13.8ul, MgCl
21.6ul, dNTPs (2.5Mm) 1.6ul, Taq enzyme (1U/ul) 1ul, 10 * damping fluid 2ul;
More preferably, the PCR reaction conditions is as follows: 94 ℃ of 2min, 94 ℃ of 20s, 56 ℃ of 30s, totally 20 circulations, 72 ℃ of 2min, 60 ℃ of 30min.
6. according to each described method in the claim 1 to 5, it is characterized in that described method steps 2) in the product that obtains of preselected amplification carry out selection amplification in the step 3) as dna profiling after through 20 times of dilutions.
7. according to each described method in the claim 1 to 5, it is characterized in that described method steps 3) in to select the amplification system of amplification be (20ul system/sample) dna profiling 4ul, primer 1ul, AFLP Core Mix 15ul;
Preferably, the PCR reaction conditions is as follows: 94 ℃ of 2min, 94 ℃ of 20s, 66 ℃ of 30s, 1 ℃ of every cycle down, totally 10 circulations, 72 ℃ of 2min, 94 ℃ of 20s, 56 ℃ of 30s, totally 20 circulations, 72 ℃ of 2min, 60 ℃ of 30min.
8. according to each described method in the claim 1 to 5, it is characterized in that described method also comprises the step that adopts sex change electrophoresis and silver staining method to detect resulting amplified production in the step 3).
9. the application of each described method in setting up Erigeron breviscapus (Vant.) Hand.-Mazz. dna molecular finger printing in the claim 1 to 8.
10. the application of each described method in genetic breeding, conservation of resources, introduction and acclimatization or the optimized varieties of Erigeron breviscapus (Vant.) Hand.-Mazz. in the claim 1 to 9.
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| CN2010102719618A CN101914629B (en) | 2010-08-31 | 2010-08-31 | Erigeron breviscapus DNA polymorphism detection method and application thereof |
| HK11100533.1A HK1146303A1 (en) | 2010-08-31 | 2011-01-20 | A method of detecting the dna polymorphism of erigeron breviscapus and the use thereof |
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|---|---|---|---|---|
| CN104145807A (en) * | 2014-07-29 | 2014-11-19 | 北京林业大学 | Method for obtaining genus hybrid of forsythia and abeliophyllum |
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2010
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2011
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Non-Patent Citations (1)
| Title |
|---|
| 《中国优秀硕士学位论文全文数据库（电子期刊）》 20070215 李玉舒 《中国玫瑰种质资源调查及其品种分类学研究》 51－68页，图5－1，表5－1 1－10 , 第2期 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104145807A (en) * | 2014-07-29 | 2014-11-19 | 北京林业大学 | Method for obtaining genus hybrid of forsythia and abeliophyllum |
| CN104145807B (en) * | 2014-07-29 | 2016-02-10 | 北京林业大学 | A kind of method obtaining Forsythia and Abelia dielsii leaf and bigener |
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| CN101914629B (en) | 2012-01-25 |
| HK1146303A1 (en) | 2011-05-27 |
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