CN104962632A - Primers for detecting B1 type mutation of soybean fatty acid dehydrogenase synthesis gene and application thereof - Google Patents
Primers for detecting B1 type mutation of soybean fatty acid dehydrogenase synthesis gene and application thereof Download PDFInfo
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- CN104962632A CN104962632A CN201510386751.6A CN201510386751A CN104962632A CN 104962632 A CN104962632 A CN 104962632A CN 201510386751 A CN201510386751 A CN 201510386751A CN 104962632 A CN104962632 A CN 104962632A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses primers for detecting B1 type mutation of a soybean fatty acid dehydrogenase synthesis gene and application thereof and belongs to the technical field of molecular marker development of plants and molecule assisted selection of crops. Nucleotide sequences of the primers provided by the invention are shown in SEQ ID No. 1 and SEQ ID No. 2. Meanwhile, the invention also provides a method for rapidly detecting the B1 type mutation of the soybean fatty acid dehydrogenase synthesis gene by using the primers. According to the method, PCR (Polymerase Chain Reaction) amplification is carried out by taking DNA (Deoxyribonucleic Acid) of a sample to be detected as a template and taking nucleotide sequences shown in SEQ ID No. 1 and SEQ ID No. 2 as the primers, then, an obtained amplification product is subjected to enzyme digestion, and finally, an enzyme digestion product is identified by electrophoresis. The primers provided by the invention and enzyme digestion identification reaction can be used for rapidly and accurately detecting a B1 type mutant site for controlling the content of high oleic acid in soybeans; and the period of screening of high-oleic-acid soybean materials can be greatly shortened, and a molecular marker resource is provided for breeding high-oleic-acid soybean varieties.
Description
Technical field
The present invention relates to a kind of for detect soybean fat acidohydrogenase synthetic gene B1 type sudden change primer and application, belong to plant molecular marker exploitation and crop molecule assisted Selection technical field.
Background technology
Soybean provides important plant oil content for the mankind, and the oleaginousness of the soybean most important economic characters that are soybean.Commodity soybean oil mainly comprises the Palmiticacid (16:0) of about 11%, the stearic acid (18:0) of 4%, the oleic acid (18:1) of 25%, the linolic acid (18:2) of 52% and the linolenic acid (18:3) (Fehr, 2007) of 4%.The natural condition such as weather, soil, water quality of China regions and breed breeding direction all there are differences, deviation creation condition is enriched for Soybean Germplasm quality characteristic, be conducive to the soybean germplasm (Gai Junyi etc., 2001) screening excellent lipid acid composition.Unsaturated fatty acids is essential fatty acid, tool in human metabolism
There is important physiological function, oleic acid effectively can reduce total cholesterol and bad cholesterol content in blood, nutritionist is referred to as " safe fats acid ", and linolic acid has suppression cancer, prevent diabetes and minimizing body fat, reduce Blood Cholesterol amount, prevent arteriosclerotic effect, linolenic acid has thrombus dissolving, hypotensive anti-platelet aggregation, the anti-ageing effect of waiting for a long time of brain tonic (Chen Xuezhen, 2004).Unsaturated fatty acids in soybean oil has important Functions of Physiological Health Care, in unsaturated fatty acids, the stability of oleic acid is best, improve its content and can extend storage period, but because linoleic acid plus linolenic acid is respectively containing 2,3 unsaturated double-bonds, easily make Oxidation of Fat and Oils go bad, cause soya-bean oil and food taste thereof not good, nutritive value reduces.In addition, because saturated fatty acid energy is low, non-digestible, too much edible can cause fat disease and cardiovascular disorder (willow etc., 2006).Therefore, improve oleic acid content, reducing linoleic acid content is one of important goal of soybean quality breeding.
In the route of synthesis of grease, fatty acid dehydrogenase 2 (FAD2) serves vital effect (Okuley etc., 1994 in the transition process of oleic acid precursor to linolic acid precursor; Schlueter etc., 2007), in the soybean seeds of growing, 2 fatty acid desaturase GmFAD2-1A (Glyma10g42470) and GmFAD2-1B (Glyma20g24530) expression level the highest, therefore these 2 genes are considered to the candidate gene controlling soybean oil acid content, and increasing investigator cultivates the high quality soybean kind of high gas oil ratio content by transforming these 2 target genes.Genetically engineered and candidate gene molecular breeding strategy are used to cultivate high quality soybean (Buhr etc., 2002 higher than 80% oleic acid content; Hoshino etc., 2010; Pham etc., 2010), the mutation content that these achievements in research demonstrate in GmFAD2-1A and GmFAD2-1B gene is more serious, and the oleic acid content in soybean oil is higher, and the stability of grease is also stronger simultaneously.
China, as the source region of soybean, has abundant Soybean Germplasm.At present, China's crop germplasm resource storehouse oneself the Soybean Germplasm preserved reach more than 30,000 parts, occupy (Wang Yuesheng, 2002) first of the world.But it is large to utilize traditional oil content authentication method to carry out qualification workload to the soybean resource of huge number, the problem that the cycle is long, still do not have at present a kind of can the method in the fast and exactly B1 type mutational site of detection control soybean high gas oil ratio content.
Summary of the invention
For solving the problem, the invention provides a kind of primer for detecting the sudden change of soybean fat acidohydrogenase synthetic gene B1 type, the technical scheme taked is as follows:
One object of the present invention is to provide a kind of primer for detecting the sudden change of soybean fat acidohydrogenase synthetic gene B1 type, and the nucleotide sequence of this primer is as shown in SEQ ID NO.1-SEQ ID NO.2.
Another object of the present invention is to provide a kind of method utilizing described primer rapid detection soybean fat acidohydrogenase synthetic gene B1 type to suddenly change, the method with the DNA of testing sample for template, arrange as primer carries out pcr amplification with the such as nucleotides sequence shown in SEQ ID NO.1-SEQ ID NO.2, cut carrying out enzyme to obtained amplified production, finally utilize electroresis appraisal digestion products.
The step of described method is as follows:
1) DNA of testing sample is extracted, for subsequent use;
2) with step 1) gained DNA is template, arranges as primer carries out pcr amplification with the such as nucleotides sequence shown in SEQ ID NO.1-SEQ ID NO.2, obtains pcr amplification product;
3) restriction endonuclease is utilized to step 2) gained pcr amplification product carries out enzyme and cuts process, obtains digestion products;
4) electroresis appraisal step 3 is utilized) gained digestion products.
Preferably, step 2) described pcr amplification, amplification condition is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 40s, and 52 DEG C of annealing 30s, 72 DEG C extend 15s, totally 40 circulations, then 72 DEG C extend 4min, 4 DEG C of insulations.
Preferably, step 3) described enzyme cuts process, and be utilize ApaI restriction endonuclease, at 37 DEG C, enzyme cuts through night.
More preferably, described enzyme is cut, and the consumption cutting ApaI restriction endonuclease in system at 10 μ l enzymes is 0.2 μ l.
Preferably, step 4) the described electrophoresis that utilizes identifies, there is B1 sudden change in the soybean fat acidohydrogenase synthetic gene of the sample containing 239bp band.
More preferably, the described electrophoresis that utilizes is identified, the sample only containing 239bp band is soybean fat acidohydrogenase synthetic gene B1 mutant homozygous body, and the sample containing 239bp and 261bp band is soybean fat acidohydrogenase synthetic gene B1 sudden change heterozygote.
The concrete steps of described method are as follows:
1) DNA of testing sample is extracted, for subsequent use;
2) with step 1) gained DNA is template, arrange as primer carries out pcr amplification with the such as nucleotides sequence shown in SEQ ID NO.1-SEQ ID NO.2, amplification condition is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 40s, 52 DEG C of annealing 30s, 72 DEG C extend 15s, totally 40 circulations, 72 DEG C extend 4min, 4 DEG C of insulations again, obtain pcr amplification product;
3) ApaI restriction endonuclease is utilized to step 2) gained pcr amplification product enzyme at 37 DEG C cuts through night, obtains digestion products;
4) electroresis appraisal step 3 is utilized) gained digestion products, there is B1 sudden change in the soybean fat acidohydrogenase synthetic gene of the sample containing 239bp band.
Described either method is used for screening and identification soybean fat acidohydrogenase synthetic gene B1 type mutant.
Beneficial effect of the present invention:
The present invention is on the soybean fat acidohydrogenase synthetic gene GmFAD2-1B basis cloned, by comparing the GmFAD2-1B gene order of high gas oil ratio soybean B1 and conventional oil acid content soybean BAY, Late Cambrian is positioned at the single base mutation (C → G) at 409bp place, and this sudden change causes oleic acid content to change.
The present invention is directed to B1 mutational site and devise 1 cover molecule marker (upstream primer FAD2-1B-F2D, downstream primer FAD2-1B-R2, as shown in SEQ ID NO.1-SEQ ID NO.2) carry out the pcr amplification comprising mutational site, and utilize endonuclease ApaI to carry out endonuclease reaction to amplified production, finally utilize polyacrylamide gel electrophoresis to carry out fragment separation to digestion products, finally identify B1 mutational site.Wherein, primer involved in the present invention and enzyme cut identification reaction can the B1 type mutational site of fast and exactly detection control soybean high gas oil ratio content, the present invention can shorten the screening cycle of high gas oil ratio soybean material greatly, and the cultivation for high gas oil ratio soybean varieties provides molecule marker resource.
Accompanying drawing explanation
Fig. 1 is GmFAD-2-1B (soybean fat acidohydrogenase synthetic gene) B1 type mutational site.
Fig. 2 identifies B1 mutational site for utilizing polyacrylamide gel electrophoresis;
(wherein identify blue arrow indication fragment and comprise B1 mutational site, Lane1 is A3 × B1-1 heterozygous individual; Lane2 is A3 × B1-2 heterozygous individual, and Lane3 is A3 × B1-3 heterozygous individual; Lane4 is A3 × B1-4 heterozygous individual; Lane5 is A3 × B1-5 heterozygous individual; Lane 6 is A3 × B1-6 heterozygous individual; Lane7 is A3 × B2 heterozygous individual; Lane10 is A3 × B1-7 heterozygous individual; Lane12 is for closing rich × B2 heterozygous individual; Lane13 is black agriculture × B1 heterozygous individual; Lane14 is middle Huang × B2-1 heterozygous individual; Lane15 is middle Huang × B2-2 heterozygous individual; LaneB1 is B1 homozygous individual; Lane34 is He3e4e7; Lane is combined into rich homozygous individual; Be middle yellow homozygous individual in Lane).
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not by the restriction of embodiment.
Material therefor, reagent, instruments and methods in following examples, without specified otherwise, be the conventional material in this area, reagent, instruments and methods, all obtain by commercial channel.
Embodiment 1
1) (NCBI ID is 100805777 first to clone the GmFAD2-1B gene order of high gas oil ratio soybean B1 and conventional oil acid content soybean BAY, soybean gene group database sequence is numbered: Glyma20g24530), DNAstar software is utilized to carry out sequence alignment Late Cambrian single base mutation (sequence alignment the results are shown in accompanying drawing 1), this mutational site occurs in the 409bp place of GmFAD-2-1B, and the C that SNP sports wild-type BAY becomes the G of A3 type.The DNA of testing sample is provided simultaneously.
2) according to step 1) in mutational site design dCAPS mark upstream primer FAD2-1B-F2D, downstream primer FAD2-1B-R2 (as shown in SEQ ID NO.1-SEQ ID NO.2), utilize the pcr amplification carrying out GmFAD2-1B mutational site using the genomic dna of material to be identified as template, the PCR reaction system (table 1) adopted and amplification program as follows:
Table 1 PCR reaction system
Pcr amplification condition is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 40s, and 52 DEG C of annealing 30s, 72 DEG C extend 15s, totally 40 circulations, then 72 DEG C extend 4min, 4 DEG C of insulations.
3) to step 2) in PCR primer carry out endonuclease reaction, getting PCR primer is template, adds 1 μ l NEB4 respectively
#buffer, 0.2 μ l ApaI restriction endonuclease, finally use 3.8 μ l ultrapure water polishings to total reaction volume 101 μ l, 37 DEG C of enzymes that spend the night are cut.
4) get step 3) in digestion products 5 μ l, add 1 μ l 6 × loading buffer, point sample carries out electrophoresis in the polyacrylamide gel loading wells prepared.
Wherein, reagent needed for electrophoresis and concrete reaction process as follows.
Experiment reagent: 30% acrylamide storage liquid (37.5:1) keeps in Dark Place; 1.5M Tris-HCl buffer (pH 8.8); 0.5M Tris-HCl buffer (pH 6.8); 10%APS (ammonium persulphate); TEMED; 1X Tris-glycine electrophoretic buffer.
Preparation separation gel: according to the separation gel of the size preparation respective volume of concrete offset plate capacity, for 10ml 12% separation gel, add distilled water 3.43ml successively; 30% acrylamide 4ml; 1.5M Tris-HCl buffer (pH 8.8) 2.5ml; 10%APS60 μ l; TEMED 13 μ l.Add 1ml water at Jiao Mianshang after encapsulating and ensure the smooth of glue face.
The concentrated glue glue of preparation: concentrate glue for 5ml 12% and add distilled water 3ml successively; 30% acrylamide 700 μ l; 1.5MTris-HCl buffer (pH 8.8) 1.25ml; 10%APS 25 μ l; TEMED 20 μ l.Comb is inserted immediately after encapsulating.
After gelling to be concentrated is admittedly good, pcr amplification product application of sample is carried out electrophoresis on non-denaturing polyacrylamide gel, deposition condition is 230V electrophoresis 2 ~ 3 hours (the concrete time is according to target fragment sizes), after electrophoresis terminates, glue is placed in EB solution dyeing 15 minutes, wash away residual EB with distilled water, imaging in DNA gel imaging system also adds up banding pattern.
5) according to step 4) in gel imaging result detect B1 mutational site, as shown in Figure 2, the individuality identifying the 239bp band of arrow indication includes B1 mutational site.The single band (band indicated by arrow) that the B1 mutated individual isozygotied is 239bp, the heterozygous individual including B1 mutational site has two band (239bp and 261bp).
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention; any person skilled in the art; not departing from spirit and scope of the invention; various changes and modification can be done; therefore, what protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1., for detecting a primer for soybean fat acidohydrogenase synthetic gene B1 type sudden change, it is characterized in that, nucleotide sequence is as shown in SEQ ID NO.1-SEQ ID NO.2.
2. one kind utilizes the method for primer rapid detection soybean fat acidohydrogenase synthetic gene B1 type sudden change described in claim 1, it is characterized in that, with the DNA of testing sample for template, arrange as primer carries out pcr amplification with the such as nucleotides sequence shown in SEQ ID NO.1-SEQ ID NO.2, cut carrying out enzyme to obtained amplified production, finally utilize electroresis appraisal digestion products.
3. method described in claim 2, is characterized in that, step is as follows:
1) DNA of testing sample is extracted, for subsequent use;
2) with step 1) gained DNA is template, arranges as primer carries out pcr amplification with the such as nucleotides sequence shown in SEQ ID NO.1-SEQ ID NO.2, obtains pcr amplification product;
3) restriction endonuclease is utilized to step 2) gained pcr amplification product carries out enzyme and cuts process, obtains digestion products;
4) electroresis appraisal step 3 is utilized) gained digestion products.
4. method described in claim 3, is characterized in that, step 2) described pcr amplification, amplification condition is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 40s, and 52 DEG C of annealing 30s, 72 DEG C extend 15s, totally 40 circulations, then 72 DEG C extend 4min, 4 DEG C of insulations.
5. method described in claim 3, is characterized in that, step 3) described enzyme cuts process, and be utilize ApaI restriction endonuclease, at 37 DEG C, enzyme cuts through night.
6. method described in claim 5, is characterized in that, described enzyme is cut, and the consumption cutting ApaI restriction endonuclease in system at 10 μ l enzymes is 0.2 μ l.
7. method described in claim 3, is characterized in that, step 4) the described electrophoresis that utilizes identifies, there is B1 sudden change in the soybean fat acidohydrogenase synthetic gene of the sample containing 239bp band.
8. method described in claim 7, it is characterized in that, the described electrophoresis that utilizes is identified, sample only containing 239bp band is soybean fat acidohydrogenase synthetic gene B1 mutant homozygous body, and the sample containing 239bp and 261bp band is the heterozygote that soybean fat acidohydrogenase synthetic gene B1 suddenlys change.
9. method described in claim 3, is characterized in that, concrete steps are as follows:
1) DNA of testing sample is extracted, for subsequent use;
2) with step 1) gained DNA is template, arrange as primer carries out pcr amplification with the such as nucleotides sequence shown in SEQ ID NO.1-SEQ ID NO.2, amplification condition is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 40s, 52 DEG C of annealing 30s, 72 DEG C extend 15s, totally 40 circulations, 72 DEG C extend 4min, 4 DEG C of insulations again, obtain pcr amplification product;
3) ApaI restriction endonuclease is utilized to step 2) gained pcr amplification product enzyme at 37 DEG C cuts through night, obtains digestion products;
4) electroresis appraisal step 3 is utilized) gained digestion products, there is B1 sudden change in the soybean fat acidohydrogenase synthetic gene of the sample containing 239bp band.
10. either method described in claim 2-9, is characterized in that, for screening and identification soybean fat acidohydrogenase synthetic gene B1 type mutant.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628481A (en) * | 2019-01-02 | 2019-04-16 | 吉林农业大学 | Soya bean fatty acid desaturase polygenes CRISPR/Cas9 vector construction and application |
| CN113999928A (en) * | 2021-10-13 | 2022-02-01 | 江苏省农业科学院 | Gene GmFAD2-1B site related to content of soybean oleic acid, InDel molecular marker and application |
| CN114731949A (en) * | 2022-03-31 | 2022-07-12 | 浙江新安化工集团股份有限公司 | High-oleic acid soybean mutant and detection method thereof |
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| US20130295253A1 (en) * | 2012-05-01 | 2013-11-07 | Susan Knowlton | Environmentally sustainable frying oils |
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| CN102753684A (en) * | 2009-06-22 | 2012-10-24 | 国立大学法人佐贺大学 | Mutant for increasing oleic acid content of soybean oil and fat, and responsible gene therefor |
| CN103687479A (en) * | 2011-01-14 | 2014-03-26 | 密苏里大学管委会 | Method to develop high oleic acid soybeans using conventional soybean breeding techniques |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628481A (en) * | 2019-01-02 | 2019-04-16 | 吉林农业大学 | Soya bean fatty acid desaturase polygenes CRISPR/Cas9 vector construction and application |
| CN113999928A (en) * | 2021-10-13 | 2022-02-01 | 江苏省农业科学院 | Gene GmFAD2-1B site related to content of soybean oleic acid, InDel molecular marker and application |
| CN113999928B (en) * | 2021-10-13 | 2023-05-26 | 江苏省农业科学院 | Gene GmFAD2-1B locus related to soybean acid content, inDel molecular marker and application |
| CN114731949A (en) * | 2022-03-31 | 2022-07-12 | 浙江新安化工集团股份有限公司 | High-oleic acid soybean mutant and detection method thereof |
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Application publication date: 20151007 |