CN1113593C - Method for quickly determining purity of hybrid seed and sterility line of hybrid rice - Google Patents
Method for quickly determining purity of hybrid seed and sterility line of hybrid rice Download PDFInfo
- Publication number
- CN1113593C CN1113593C CN 99103138 CN99103138A CN1113593C CN 1113593 C CN1113593 C CN 1113593C CN 99103138 CN99103138 CN 99103138 CN 99103138 A CN99103138 A CN 99103138A CN 1113593 C CN1113593 C CN 1113593C
- Authority
- CN
- China
- Prior art keywords
- seed
- minutes
- sample
- electrophoresis
- gel
- 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a method for rapidly and exactly identifying the purity of sterile-line seeds and the purity of hybrid seeds of hybrid rice, which comprises seed treatment, DNA sample preparation, polymerase chain reaction, polyacrylamide gel electrophoresis, silver nitrate dyeing and result observation. Compared with the existing identifying method for the purity of seeds, the identifying method for the purity of seeds of the present invention has the advantages of short time, high speed, high precision, low cost, etc.
Description
Technical field:
The present invention relates to indica hybrid rice male sterile line and purity of hybrid authentication method, belong to crop seeds purity authenticate technology field.
Technical background:
The successful utilization of China hybrid rice on producing is highly visible, and China's grain-production is made a great contribution.In recent years, average annual cultivated area all meets or exceeds more than 50% of paddy rice cultivated area.On hybrid rice produced, hybrid seed purity was directly connected to the performance of hybrid rice yield potentiality, is related to its promotion and application.In sterile line propagation, maintenance line usually mixes in the male-sterile seed of producing.Because male sterile line and maintenance line belong to the genetic affinity with dyskaryosis, extremely difficulty is distinguished it with conventional method and means.In hybrid seed is produced, because the large tracts of land production of hybrid seeds, usually can not be in time, the maintenance line that will mix in male sterile line up hill and dale removes, mix in hybrid seed.Simultaneously, when the results crossbreed, the part spike of rice of recovery system is often sneaked in the crossbreed and is mixed.Because the above-mentioned phenomenon that mixes is difficult to avoid, male-sterile seed and hybrid seed purity all need to carry out strict evaluation; Just can guarantee production application.
Identify problem for the purity that solves " three are " sterile line propagation and hybrid seeding, all carried out some researchs and exploration both at home and abroad, but failed to find a kind of authentication method of hybrid seed purity fast and accurately eventually.In country's " the Seventh Five-Year Plan " paddy rice emphasis tackling key problem, hybrid rice center, Hunan utilizes fluorescence detection that hybrid seed purity is detected, but the reliability, the accuracy that detect all do not reach requirement far away; Also there is the scholar to utilize the specific band spectrum of DNA analysis technology pair cell nuclear gene group to distinguish evaluation, need at least two above molecular labelings, often obscure based on the discriminating of nuclear gene in addition, some uncertain factors occur, and cost is higher to the material of similar genetic background.Nonetheless, it also is at a loss what to do for the purity of sterile line propagation.Up to the present, unique method is exactly male-sterile seed or the hybrid seed that will produce, identifies through the sampling plantation that Hainan Island carries out purity of taking.This method is bothersome, effort, time-consuming, and has a strong impact on the plan and the sale of seed.This has been that at different levels kinds of subdivisions were expected one of important technology difficult problem that solves since hybrid rice was promoted.
Summary of the invention:
Purpose of the present invention is intended to obtain a kind of effective ways, the male-sterile seed of breeding is made a distinction with the maintenance line that mixes: hybrid seed is detected with maintenance line that mixes and recovery system, thereby identify the purity of male-sterile seed or hybrid seed quickly and accurately, bring into play hybrid rice hybrid vigour potentiality to greatest extent.
The invention is characterized in according to the cytoplasm of male-sterile seed or hybrid seed and the cytoplasm of its maintenance line and recovery system and have certain difference, comprise DNA and protein difference, and by a primer with unique dna sequence, utilize modern archaeal dna polymerase chain reaction (Polymerase Chain Reaction) technology, the purity of male-sterile seed or hybrid seed is quick, accurately discriminating.Its detailed authentication method is as follows:
A kind of method of fast, accurately identifying sterile line of hybridized rice seed and hybrid seed purity.Comprise seed treatment, DNA sample preparation, polymerase chain reaction, polyacrylamide gel electrophoresis, cma staining, observational technique as a result.
(1) seed treatment: rice sterile line or crossbreed seed are placed under 25 ℃ of conditions, soaked seed 48 hours; Change in the culture dish, 32 ℃ are carried out moistening vernalization 24 hours again, cultivate 3-4 days for 25 ℃;
(2) DNA sample preparation: young shoot and the tender leaf with every paddy takes off respectively, is cut into 0.5 centimeter length segment, puts into and grinds cylinder, adds 250ul DNA extraction liquid, is milled into homogenate; Add 250ul DNA extraction liquid mixing again, change the 1.5ml centrifuge tube over to, adding volume ratio is 24: 1 chloroform/isoamyl alcohol 300ul, vibrates after 5 minutes, with centrifugal 30 seconds of 3000 rev/mins of rotating speeds, supernatant is changed in another 1.5ml centrifuge tube, after adding 500ul 100% freezing ethanol mixes, left standstill 5 minutes, removed supernatant in centrifugal again 1 minute, add 400ul 70% alcohol flushing 2 times or/and after centrifugal 20 seconds, remove ethanol, drying, be dissolved in the 30ulTE buffer solution stand-by;
(3) polymerase chain reaction: the DNA sample that 2ul is extracted adds in the 0.5ml centrifuge tube, add the 8ul reactant liquor again after, add half dropstone wax oil, carry out the PCR circular response, reaction cycle is followed successively by: circulation in 94 ℃/2 minutes 30 seconds 1 time; 94 ℃/40 seconds, 53 ℃/1 minute, circulation in 72 ℃/1 minute and 30 seconds 35 times; 72 ℃/8 minutes; At last, 5ul is gone up reactant liquor at 3 times of ends add the abundant mixing of 0.5ml reaction centrifuge tube, it is stand-by to put into-20 ℃ of storages;
(4) polyacrylamide gel electrophoresis: large and small each electrophoresis glass of one is cleaned with distilled water, cleaned with 95% ethanol again, evenly embrocate fritter electrophoresis glass, after dry 5 minutes, wipe unnecessary silication agent with 95% ethanol with the silication agent of fresh configuration; Evenly embrocate bulk electrophoresis glass with several Sigma-cote, dry 5 minutes; Two electrophoresis glass of size are stacked, and put into the 0.4mm spacer shims between them, it is poor to form gel with wide adhesive tape sealing, clamps with big ticket-holder all around; Preparation 4%70ml denaturing polyacrylamide gel: with 29.4g urea and 5 times of TBE of 7.0ml, heated and stirred to urea dissolves, and adds the 7.0ml40% acrylamide, and is fixed molten to 70.0ml with distilled water again, puts into ice and cool off; Add behind 700ul 10% ammonium persulfate of new preparation and the quick mixing of 70ulTEMED behind the encapsulating, immediately the flat side skewer of 0.4mm plastic comb is gone in the glue, the degree of depth is about 7mm, place after at least 4 hours, plastic comb is taken out, remove the ticket-holder and the adhesive tape at edge, with gel pack into electrophoresis poor in, add 0.5 times of TBE electrophoresis liquid, and wash the gel top 3--4 time with electrophoresis liquid, skewer is gone into upward sample comb of 0.4mm again; Sample is placed the PCR instrument, 95 ℃ of 5 minutes degenerative treatments, each sample is got in the 8.0ul adding in the sample hole, carries out the voltage stabilizing electrophoresis under 40-50V/cm, 1 hour 30 minutes time;
(5) cma staining: reagent is prepared: fixative 1500ml, 150ml glacial acetic acid wherein, 150ml methyl alcohol, 1200ml distilled water; Dyeing liquor 2000ml, 2.0g silver nitrate wherein, 3.0ml 37% formalin, 1997ml redistilled water; Flushing liquor 2000ml, 60.0g sodium carbonate wherein, 3.0ml 37% formalin, 400ul, 10mg/ml sodium thiosulfate, 1997ml distilled water; Operating process: take off the running gel plate, lie against on the table, take out spacer shims and last sample comb, go into the lower right corner, carefully firmly lever up big glass, make it and gel separation, and take off big glass with the single-edge blade skewer; The little glass handle with care that will have a gel is put into fixer and is fixed 20 minutes, puts into the distilled water wash secondary, and each 2 minutes, change in the dyeing liquor, dyeed 30 minutes; With 10 seconds of distilled water wash, change in the flushing liquor, washed 5--10 minute; Change in the fixative fixedly 5--6 minute over to; At last, use distilled water wash 2-5 minute;
(6) result observes: the gel glass plate after will dyeing is placed under the fluorescent lamp, observes through the DNA of pcr amplification dyeing band spectrum; The sample that presents positive reaction is a male-sterile seed, and the sample of negative reaction is other accessory seed; The sample that presents two positive reactions is a hybrid seed, and the sample of single positive reaction and negative reaction is other accessory seed.
Seed purity identification method of the present invention is compared with existing seed purity identification method, and its advantage and good effect are:
1, time weak point, speed is fast.Adopt the technology of the present invention to identify rice sterile line and paddy rice cross breeding seed purity, than the field planting authentication method that seed enterprise adopts, the time is short, and speed is fast, and does not miss farming season.After the rice paddy seed results, adopt the present invention to identify seed purity in 15 days in the laboratory, purity plantation evaluation is carried out in the Hainan Island that need not to sample to, can not influence production and sales.
2, accuracy height, cost is low.Test shows, adopts the technology of the present invention to identify rice sterile line and hybrid seed purity, and than the accuracy height of fluorescence detection, reliability reaches 99.9%.Than the low 2-3 of conventional DNA analysis technical costs doubly.
Embodiment:
One, detected seed requires:
Male-sterile seed or hybrid seed should have normal percentage of seedgermination.
Two, seed sprouting is handled:
Seed is placed under 25 ℃ of conditions, soaked seed 48 hours: change in the culture dish, 32 ℃ are carried out moistening vernalization 24 hours again, cultivate 3-4 days for 25 ℃.
Three, DNA sample preparation:
Respectively the young shoot of every paddy and tender are taken off, be cut into 0.5 centimeter length segment, put into and grind cylinder, (composition: 50mMTris-HClpH8.0.25mMEDTA, 700mMNaCl 1%SDS), are milled into homogenate to add the 250ulDNA extract.Add 250ulDNA extract mixing again, change the 1.5ml centrifuge tube over to, add 300ul chloroform/isoamyl alcohol (500: 21), vibrated 5 minutes, and followed centrifugal 30 seconds (3000 rev/mins), supernatant is carefully changed in another 1.5ml centrifuge tube, after adding the freezing ethanol mixing of 500ul100%, left standstill 5 minutes, removed supernatant, and added 400ul70% alcohol flushing 2 times (in case of necessity can be centrifugal 20 seconds) in centrifugal again 1 minute.Then, remove ethanol, drying, be dissolved in the 30ulTE buffer solution (composition: 0.01mMEDTA and 10mMTris-Cl, pH7.6) in, stand-by.
Four; Polymerase chain reaction:
The DNA sample that 2ul is extracted adds in the 0.5ml centrifuge tube, add 8ul reactant liquor (composition: lxbuffer again, 1.5mMMgCl2,200uMdNTP, 0.1uM Primer F, 0.1uM Primer R and 0.1unitTaq DNA Polymerase, to the purity of hybrid seed, need add corresponding a recovery again is labeled primer).Then, add half dropstone wax oil, carry out the PCR circular response, reaction cycle is followed successively by: 94 ℃/2 ' 30 ' ' circulate 1 time; 94 ℃/40 ', 53 ℃/1 ', 72 ℃/1 ' 30 " circulate 35 times; 72 ℃/8 '; At last, with 10ul 3X cessation reaction liquid (composition: 10mMNaOH, 95%Formamide, 0.05%Bromophenol blue, 0.05%Xylene Cyanolp 10mMEDTA) adds the abundant mixing of 0.5ml reaction centrifuge tube, puts into-20 ℃ of storages, stand-by.
Five, polyacrylamide gel electrophoresis:
One width of cloth (large and small each one) electrophoresis glass is wiped with distilled water and earned, cleans with 95% ethanol.Fritter electrophoresis glass is evenly embrocated in silication agent (1ul Binding Silane adds 1.0ml95% ethanol and 5.0ul glacial acetic acid) with fresh configuration.After dry 5 minutes, wipe unnecessary silication agent with 95% ethanol.Evenly embrocate bulk electrophoresis glass with several Sigma-cote, dry 5 minutes.Two electrophoresis glass of size are stacked, and put into 0.4mm spacer shims (about each a slice) between them, it is poor to form gel with wide adhesive tape sealing, all around with big ticket-holder clamping.Preparation 4%70ml denaturing polyacrylamide gel: 29.4g urea and 7.0ml5 * TBE (are contained 54g Tris-base in every liter of solution, 27.5g Boric acid, 20ml 0.5M EDTA, pH8.0) heated and stirred is mixed, dissolve to urea, adding 7.0ml 40% acrylamide (composition: 2%Bis-acrylamide, 38%Acrylamid).Fixed molten with distilled water again to 70.0ml, put into ice and cool off.Encapsulating behind 700ul 10% ammonium persulfate of the new preparation of adding and the quick mixing of 70ulTEMED.Behind the encapsulating, immediately the flat side skewer of 0.4mm plastic comb is gone in the glue, the degree of depth is about 7mm.Place after at least 4 hours, plastic comb is taken out, remove the ticket-holder and the adhesive tape at edge, during the electrophoresis of will packing into fixed attention is poor, adds 0.5 * TBE electrophoresis liquid, and wash the gel top 3--4 time with electrophoresis liquid, skewer is gone into upward sample comb of 0.4mm again.Sample is placed the PCR instrument, 95 ℃ of 5 minutes degenerative treatments, each sample is got in the 8.0ul adding in the sample hole, carries out voltage stabilizing electrophoresis (40-50V/cm), about 1 hour 30 minutes time.
Six, cma staining:
Reagent is prepared
Fixative: 1500ml (10%aceticacid)
The 150ml glacial acetic acid
150ml methyl alcohol
3.0ml formaldehyde
1200ml distilled water
Dyeing liquor: 20000ml
2.0g silver nitrate (severe toxicity) Silver Nitrate
3.0ml 37% formalin Formaldehyde
1997ml d distilled water (high-purity distilled water)
Flushing liquor: 2000ml (being chilled to 10 ℃ summer in advance)
60.0g sodium carbonate
3.0ml 37% formalin
400ul, 10mg/ml sodium thiosulfate
1997ml distilled water
Operating process:
Take off the running gel plate, lie against table and go up (big glass is last, and little glass is following), take out spacer shims and last sample comb, go into the lower right corner, carefully firmly lever up big glass, make it and gel separation, and take off big glass with the single-edge blade skewer.The little glass handle with care that will have a gel is put into fixer and is fixed 20 minutes, puts into the distilled water wash secondary, each 2 minutes.Change in the dyeing liquor, dyeed 30 minutes.With 10 seconds of distilled water wash, change over to (10 ℃) in the flushing liquor, washed 5-10 minute.Change in the fixative fixedly 5-4 minute over to.At last, use distilled water wash 2--5 minute.
Seven, the result observes and data statistics:
Gel glass plate after the dyeing is placed under the fluorescent lamp, observes through the DNA of pcr amplification dyeing band spectrum.Purity to sterile line propagation identifies that the sample that presents positive reaction is a male-sterile seed, and the sample of negative reaction is other accessory seed.Purity to hybrid seed identifies that the sample that presents two positive reactions is a hybrid seed, and the sample of single positive reaction and negative reaction is other accessory seed.
Claims (1)
1, a kind of method of fast, accurately identifying sterile line of hybridized rice seed and hybrid seed purity is characterized in that operating procedure is:
(1) seed treatment: rice sterile line or crossbreed seed are placed under 25 ℃ of conditions, soaked seed 48 hours: change in the culture dish, 32 ℃ are carried out moistening vernalization 24 hours again, cultivate 3-4 days for 25 ℃;
(2) DNA sample preparation: young shoot and the tender leaf with every paddy takes off respectively, is cut into 0.5 centimeter length segment, puts into and grinds cylinder, adds 250ul DNA extraction liquid, is milled into homogenate; Add 250ul DNA extraction liquid mixing again, change the 1.5ml centrifuge tube over to, adding volume ratio is 24: 1 chloroform/isoamyl alcohol 300ul, vibrates after 5 minutes, with centrifugal 30 seconds of 3000 rev/mins of rotating speeds, supernatant is changed in another 1.5ml centrifuge tube, after adding 500ul 100% freezing ethanol mixes, left standstill 5 minutes, removed supernatant in centrifugal again 1 minute, add 400ul 70% alcohol flushing 2 times or/and after centrifugal 20 seconds, remove ethanol, drying, be dissolved in the 30ulTE buffer solution stand-by;
(3) polymerase chain reaction: the DNA sample that 2ul is extracted adds in the 0.5ml centrifuge tube, add the 8ul reactant liquor again after, add half dropstone wax oil, carry out the PCR circular response, reaction cycle is followed successively by: circulation in 94 ℃/2 minutes 30 seconds 1 time; 94 ℃/40 seconds, 53 ℃/1 minute, circulation in 72 ℃/1 minute and 30 seconds 35 times; 72 ℃/8 minutes; At last, 3 times of cessation reaction liquid of 5ul are added the abundant mixing of 0.5ml reaction centrifuge tube, it is stand-by to put into-20 ℃ of storages;
(4) polyacrylamide gel electrophoresis: large and small each electrophoresis glass of one is cleaned with distilled water, cleaned with 95% ethanol again, evenly embrocate fritter electrophoresis glass, after dry 5 minutes, wipe unnecessary silication agent with 95% ethanol with the silication agent of fresh configuration; Evenly embrocate bulk electrophoresis glass with several Sigma-cote, dry 5 minutes; Two electrophoresis glass of size are stacked, and put into the 0.4mm spacer shims between them, it is poor to form gel with wide adhesive tape sealing, clamps with big ticket-holder all around; Preparation 4%70ml denaturing polyacrylamide gel: with 29.4g urea and 5 times of TBE of 7.0ml, heated and stirred to urea dissolves, and adds the 7.0ml40% acrylamide, and is fixed molten to 70.0ml with distilled water again, puts into ice and cool off; Add behind 700ul 10% ammonium persulfate of new preparation and the quick mixing of 70ul TEMED behind the encapsulating, immediately the flat side skewer of 0.4mm plastic comb is gone in the glue, the degree of depth is about 7mm, place after at least 4 hours, plastic comb is taken out, remove the ticket-holder and the adhesive tape at edge, with gel pack into electrophoresis poor in, add 0.5 times of TBE electrophoresis liquid, and wash the gel top 3--4 time with electrophoresis liquid, skewer is gone into upward sample comb of 0.4mm again; Sample is placed the PCR instrument, 95 ℃ of 5 minutes degenerative treatments, each sample is got in the 8.0ul adding in the sample hole, carries out the voltage stabilizing electrophoresis under 40-50V/cm, 1 hour 30 minutes time;
(5) cma staining: reagent is prepared: fixative 1500ml, 150ml glacial acetic acid wherein, 150ml methyl alcohol, 1200ml distilled water; Dyeing liquor 2000ml, 2.0g silver nitrate wherein, 3.0ml 37% formalin, 1997ml redistilled water; Flushing liquor 2000ml, 60.0g sodium carbonate wherein, 3.0ml 37% formalin, 400ul10mg/ml sodium thiosulfate, 1997ml distilled water; Operating process: take off the running gel plate, lie against on the table, take out spacer shims and last sample comb, go into the lower right corner, carefully firmly lever up big glass, make it and gel separation, and take off big glass with the single-edge blade skewer; The little glass handle with care that will have a gel is put into fixer and is fixed 20 minutes, puts into the distilled water wash secondary, and each 2 minutes, change in the dyeing liquor, dyeed 30 minutes; With 10 seconds of distilled water wash, change in the flushing liquor, washed 5--10 minute; Change in the fixative fixedly 5--6 minute over to; At last, use distilled water wash 2-5 minute;
(6) result observes: the gel glass plate after will dyeing is placed under the fluorescent lamp, observes through the DNA of pcr amplification dyeing band spectrum; The sample that presents positive reaction is a male-sterile seed, and the sample of negative reaction is other accessory seed; The sample that presents two positive reactions is a hybrid seed, and the sample of single positive reaction and negative reaction is other accessory seed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99103138 CN1113593C (en) | 1999-03-23 | 1999-03-23 | Method for quickly determining purity of hybrid seed and sterility line of hybrid rice |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99103138 CN1113593C (en) | 1999-03-23 | 1999-03-23 | Method for quickly determining purity of hybrid seed and sterility line of hybrid rice |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1226375A CN1226375A (en) | 1999-08-25 |
| CN1113593C true CN1113593C (en) | 2003-07-09 |
Family
ID=5271136
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99103138 Expired - Fee Related CN1113593C (en) | 1999-03-23 | 1999-03-23 | Method for quickly determining purity of hybrid seed and sterility line of hybrid rice |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1113593C (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100389209C (en) * | 2006-05-22 | 2008-05-21 | 江苏明天种业科技有限公司 | Primer for detecting seed purity and its method |
| CN101654703B (en) * | 2009-09-18 | 2012-08-22 | 云南农业大学 | Method for identifying paddy female sterile gene FST molecule |
| CN103120051A (en) * | 2013-03-05 | 2013-05-29 | 曹延明 | Rice sprouting method |
| CN103314755B (en) * | 2013-06-26 | 2015-05-06 | 江苏沿海地区农业科学研究所 | Method of detecting purity of photo-thermo genic male sterile NaCl 582S seeds |
-
1999
- 1999-03-23 CN CN 99103138 patent/CN1113593C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1226375A (en) | 1999-08-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103409553B (en) | A kind of gene chip for high-pass typing detection human papillomavirus, reagent and test kit thereof | |
| CN104059971A (en) | SSR molecular marker method of brassica allohexaploid and primers thereof | |
| CN103820320B (en) | Liquid protected by a kind of non-jelly type RNA | |
| CN109679946B (en) | Blood virus RNA protective agent and blood collection tube | |
| CN106226530A (en) | The thalline preprocess method identified for MALDI TOF antibacterial and yeast-like fungi | |
| CN1113593C (en) | Method for quickly determining purity of hybrid seed and sterility line of hybrid rice | |
| CN103184286B (en) | Identification method of rice bacterial leaf blight resistance | |
| CN101182344A (en) | Method for extracting and purifying DNA of plants like cactus | |
| CN104805179A (en) | Cabbage type rape grain weight-associated molecular marker and preparation method and application thereof | |
| CN104152547B (en) | The SSR marker finger-print of a kind of asparagus green grass or young crops standing grain bacterial classification and application | |
| CN105432598A (en) | Liquid-based cell preservation liquid and preparation method therefor | |
| CN103436618A (en) | Aphis gossypii Glover specificity SS-COI primers, kit containing primer and detection method of kit | |
| CN1928063A (en) | High-density array type cell slide culture apparatus, storage apparatus and experimental apparatus | |
| CN103609439A (en) | Influence and application method of different strains on hairy roots of ginseng | |
| CN107722121A (en) | Bee larva bacillus PLMP resists more and its application in immunochromatography paper | |
| CN103468677B (en) | Specific SS-COI (Species-specific Cytochrome Oxidase I) primers of aphis craccivora, kit containing primers and detecting method of kit | |
| CN107267498A (en) | DNA kit and method is extracted in a kind of universal material from trace plant | |
| CN110904095B (en) | Method for improving small fragment nucleic acid purification yield | |
| CN111454938A (en) | Efficient extraction method of mangrove genome DNA | |
| CN110551825A (en) | Specific primer of gulf scallop microsatellite marker and construction method and application thereof | |
| CN105063020A (en) | Method for extracting mtDNA (MiTochondrial DeoxyriboNucleic Acid) by virtue of root tissues of rape CMS (Cytoplasmic Male Sterility) system | |
| Rogers | Protein polymorphism, genic heterozygosity and divergence in the toads Bufo cognatus and B. speciosus | |
| CN1164739C (en) | Cell freeze-storing liquor for raising anabiosis rate of freeze-stored cell | |
| CN103399076A (en) | Method for identifying purity of corn seeds | |
| CN114018919B (en) | Method for detecting activity of diploid banana pollen |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |