CN103276068A - Marking primer and method for identifying channa argus and channa maculate as well as F1-generation channa argus*channa maculate and channa maculate*channa argus hybridized by channa argus and channa maculate - Google Patents
Marking primer and method for identifying channa argus and channa maculate as well as F1-generation channa argus*channa maculate and channa maculate*channa argus hybridized by channa argus and channa maculate Download PDFInfo
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Abstract
The invention discloses a marking primer for identifying channa argus and channa maculate as well as F1-generation channa argus*channa maculate and channa maculate*channa argus hybridized by the channa argus and the channa maculate on the basis of a mitochondrial DNA (deoxyribonucleic acid) sequence. The marking primer is BY14, and the marking primer BY14 consists of a left-end primer sequence BY14F and a right-end primer sequence BY14R, wherein the left-end primer sequence BY14F is 5'-CCCGTCACCCTCACCTAA-3', and the right-end primer sequence BY14R is 5'-GCGTGGGGCTACCTGTTC-3'. The invention further discloses a method for identifying the channa argus and the channa maculate as well as the F1-generation channa argus*channa maculate and channa maculate*channa argus hybridized by the channa argus and the channa maculate by using the marking primer. The marking primer can be used for rapidly identifying the channa argus and channa maculate as well as the F1-generation channa argus*channa maculate and channa maculate*channa argus hybridized by the channa argus and the channa maculate.
Description
Technical field
The present invention is specifically related to a kind of labeled primer and method of identifying snakehead, snakehead and first familiar generation spot snakehead thereof, black snakehead.
Background technology
Snakehead (
Channa argus) and snakehead (
Channa maculata) belong to together Perciformes (Perciformes), murrel suborder (Channidae), murrel section (Channidae), Ophiocephalus (
Channa).Snakehead mainly is distributed in the Changjiang river and each water system to the north of the Changjiang river, and snakehead mainly is distributed in the Zhujiang River and each water system of Hainan.In the diet tradition, these two kinds of fishes always are the objects that people like, so market demand is very big, have expedited the emergence of the aquaculture industry of murrel section fish.Nearly 10 years, the cultured area of murrel constantly enlarged, and cultured output climbs up and up.To 2011, the breed ultimate production of Chinese murrel section fish reached 44.6 ten thousand tons, and snakehead and snakehead have become one of important cultured freshwater fish of China.
The Chinese scholar better develops murrel section fish, and having researched and developed with snakehead, snakehead is parent's Hybrid, i.e. spot snakehead (snakehead ♀ * snakehead ♂) and black snakehead (snakehead ♀ * snakehead ♂).Two Hybrids all have tangible hybrid vigour than the parent, are in particular in that growth is fast, aspects such as output is high, disease is few, bait utilization height.Because cross-fertilize seed has outstanding culturing economic performance, the raiser more is ready to select cross-fertilize seed to culture.Hybridized snakehead fish has accounted for the overwhelming majority of murrel section fish breed amount at present, becomes breed variety main in the murrel section.
Snakehead, snakehead and two cross-fertilize seed crow snakeheads (snakehead ♀ * snakehead ♂), spot snakeheads (snakehead ♀ * snakehead ♂) are propagated kind artificially, can distinguish easily and differentiate snakehead, snakehead and cross-fertilize seed by morphology, AFLP molecule marker, microsatellite marker etc.But on still being the nuclear gene molecule marker, form all is difficult between two cross-fertilize seed differentiate.In production practice, it is found that black snakehead (snakehead ♀ * snakehead ♂) compares with spot snakehead (snakehead ♀ * snakehead ♂), have better growth and economic performance, as characteristics such as fast growth, efficiency of feed utilization height, cold performance be good.As propagating kind artificially, nearly all breeding enterprise all needs a kind identity validation program.How to differentiate two these problems of cross-fertilize seed in order to solve, people have carried out various trials.We recognize, the nuclear genetic material of normal cross-fertilize seed, and half is from male parent, and half is from female parent.Make that like this hybridized snakehead fish of Orthogonal Composite is identical with the nuclear genetic material source of the hybridized snakehead fish of reciprocal cross combination, utilize the molecule marker of nuclear DNA can't distinguish two cross-fertilize seed.And Mitochondrial DNA lacks gene recombination, strictly observes characteristics such as matrilinear inheritance, and the Mitochondrial DNA of first familiar generation should be from its female parent, and the kind of cross-fertilize seed just can be judged in the source that is tested and appraised Mitochondrial DNA.
Summary of the invention
The object of the present invention is to provide and a kind ofly identify the labeled primer of snakehead, snakehead and first familiar generation spot snakehead thereof, black snakehead based on mtdna sequence, this labeled primer can PCR method Rapid identification snakehead, snakehead and first familiar generation spot snakehead and black snakehead.
The present invention also aims to provide a kind of utilizes above-mentioned labeled primer based on mtdna sequence evaluation snakehead, snakehead and first familiar generation spot snakehead thereof, black snakehead to identify the method for snakehead, snakehead and first familiar generation spot snakehead thereof, black snakehead.
First purpose of the present invention is achieved by the following technical solution: a kind of labeled primer of identifying snakehead, snakehead and first familiar generation spot snakehead and black snakehead based on mtdna sequence, described labeled primer is BY14, described labeled primer BY14 is made up of left end primer sequence BY14F and right-hand member primer sequence BY14R, wherein said left end primer sequence BY14F is: 5 '-CCCGTCACCCTCACCTAA-3 ', right-hand member primer sequence BY14R is: 5 '-GCGTGGGGCTACCTGTTC-3 '.
Second purpose of the present invention is achieved by the following technical solution: a kind of utilization above-mentionedly identifies that based on mtdna sequence the labeled primer of snakehead, snakehead and first familiar generation spot snakehead and black snakehead identifies the method for snakehead, snakehead and first familiar generation spot snakehead and black snakehead, utilize above-mentioned labeled primer, amplification snakehead, snakehead, spot snakehead and black snakehead, wherein snakehead and spot snakehead have the PCR specific band to occur at the 201bp place, snakehead and black snakehead then do not have band to occur, thereby identify spot snakehead and black snakehead.
Labeled primer described in the present invention obtains by the following method:
(1) extract the complete genome DNA of snakehead, snakehead, spot snakehead and black snakehead, and measure the Mitochondrial DNA complete sequence of snakehead, snakehead, spot snakehead and black snakehead, with reference to allele-specific PCR principle, the design labeled primer;
(2) labeled primer with design carries out pcr amplification, and the electrophoresis result of contrast snakehead, snakehead, spot snakehead and 4 kinds of fishes of black snakehead judges tentatively whether this labeled primer can be utilized;
(3) the pcr amplification product purifying is reclaimed, order-checking is compared sequencing result with snakehead and spot snakehead, judges whether the dna fragmentation that pcr amplification obtains is synthesized as template by mtDNA;
(4) labeled primer is carried out pcr amplification to a plurality of snakeheads, snakehead, spot snakehead and black snakehead sample, electrophoresis then, whether the labeled primer of observing design can normally act in great amount of samples, judge the application feasibility of primer, wherein snakehead group and spot snakehead group has the PCR specific band at about 200bp place, and snakehead group and black snakehead group do not have band to occur, and this labeled primer has specificity, can be as the labeled primer of identifying spot snakehead and black snakehead.
Wherein in the step of the present invention (4) in a plurality of snakeheads, snakehead, spot snakehead and the black snakehead sample every kind of fish have 30 samples at least.
The present invention has following advantage:
(1) the Mitochondrial DNA sequencing result of snakehead, snakehead, black snakehead (snakehead ♀ * snakehead ♂), 4 kinds of fishes of spot snakehead (snakehead ♀ * snakehead ♂) explanation Mitochondrial DNA is strict with matrocliny really among the present invention, and the filial generation Mitochondrial DNA comes from female parent;
(2) design of primers that the present invention is based on mtdna sequence can be got rid of the influence of nuclear gene group DNA, can deal with problems very easily;
(3) the present invention considers accuracy and terseness, and this experiment selects for use at least 30 samples of every kind of fish to verify.The PCR of every group of 30 fishes of process detects, and electrophoresis result shows that 30 fishes of snakehead group and spot snakehead group can both amplify the purpose band, and snakehead group and black snakehead group then do not have band, and this presentation of results Auele Specific Primer has the specificity of planting;
(3) though authentication method of the present invention utilizes is the mtDNA mark, but not needing to extract separately mtDNA identifies, a small amount of mtDNA in the conventional full genome that extracts, can utilize primer to pass through pcr amplification, just can access the result behind the electrophoresis, cycle time is short, and method simply can be carried out in enormous quantities, can obtain the result in one day;
(4) the present invention can simply tell the difference of two cross-fertilize seed for differentiating that by molecular method the cross-fertilize seed of reciprocal cross provides a kind of method later at the Auele Specific Primer that mtDNA designs, and two parents also can use for Rapid identification.
Description of drawings
Fig. 1 is the electrophorogram of labeled primer BY14PCR amplified production in the embodiment of the invention 2;
Fig. 2 is that sequencing result after amplified production reclaims in the embodiment of the invention 2 and the Mitochondrial DNA of snakehead carry out blast comparison result figure.
Embodiment
The present invention is further elaborated below in conjunction with accompanying drawing, but content of the present invention is not limited thereto.
Embodiment 1
The labeled primer of identifying snakehead, snakehead and first familiar generation spot snakehead and black snakehead based on mtdna sequence provided by the invention, this labeled primer is BY14, labeled primer BY14 is made up of left end primer sequence BY14F and right-hand member primer sequence BY14R, wherein said left end primer sequence BY14F is: 5 '-CCCGTCACCCTCACCTAA-3 ', right-hand member primer sequence BY14R is: 5 '-GCGTGGGGCTACCTGTTC-3 '.
Embodiment 2
The provided by the invention utilization identifies that based on mtdna sequence the labeled primer of snakehead, snakehead and first familiar generation spot snakehead and black snakehead identifies the method for spot snakehead and black snakehead among the embodiment 1, utilize the labeled primer among the embodiment 1, amplification snakehead, snakehead, spot snakehead and black snakehead, wherein snakehead and spot snakehead have the PCR specific band to occur at the 201bp place, snakehead and black snakehead then do not have band to occur, thereby identify spot snakehead and black snakehead.
Labeled primer among the present invention obtains by the following method:
(1) get each 30 tail fish of snakehead, snakehead, spot snakehead (snakehead * snakehead), black snakehead (snakehead * snakehead), clip fin ray tissue, it is ℃ standby to be kept at 95% ethanol-20.Adopt this area ordinary method to extract the complete genome DNA of 4 kinds of fishes, be kept at-20 ℃ standby.
(2) measured the Mitochondrial DNA complete sequence (sequence is specifically seen Genbank database serial number KC823606, KC823605, KC823604, KC823607) of snakehead, snakehead, spot snakehead, black snakehead.Carry out the mtDNA comparison of 4 kinds of fishes by clustalX2.1, hybrid generation is very close with its maternal mtDNA.On the otherness sequence of two hybrid generations, we according to snakehead mtDNA with Primer 5. 0 software designs Auele Specific Primer be labeled primer, this labeled primer is F:5 '-CCCGTCACCCTCACCTAA-3 ', R:5 '-GCGTGGGGCTACCTGTTC-3 '.The principle of design of primers is with reference to allele specific pcr (allele specific PCR), because the difference of mtDNA, snakehead and black snakehead can't carry out normal pcr amplification, and snakehead and spot snakehead can, just can judge the classification of detected fish according to the electrophoresis based on DNA of this area routine.
(3) primer screening
Primer with design carries out pcr amplification respectively, and whether the electrophoresis result of 4 kinds of fishes of contrast conforms to designed size according to the size of principle of design and PCR product, judges tentatively whether primer can be utilized.The pcr amplification reaction volume is 20 μ L, and the loop parameter of PCR reaction is: 94 ℃ of sex change 30s, and 55 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations, 72 ℃ are extended 10min.Detect amplification, gel imaging instrument sweep record result with 1.0% agarose gel electrophoresis.The PCR reaction soln adopts the reaction soln of commercially available routine to get final product.
(4) different primer amplification sequencing fragment checking
The pcr amplification product purifying is reclaimed the PMD that the DNA that reclaims is connected to
TMThe 18-T carrier is selected and is checked order after positive colony detects.
Sequencing result is compared with snakehead and spot snakehead, judges whether the dna fragmentation that pcr amplification obtains is synthesized as template by mtDNA.
(5) checking of different primer in 4 kinds of fishes
The primer of acquisition preliminary screening, further checking, every kind of fish adopts 30 samples to carry out pcr amplification, and whether electrophoresis is observed the primer that screens and can normally be acted in great amount of samples then, judges the application feasibility of primer.
(6) result
6.1 the result shows, the labeled primer BY14 of the present invention's design can amplify the identical product of size at snakehead with spot snakehead group, all do not have amplified production snakehead and spot snakehead group, judge that from electrophorogram the product size also conforms to expection, concrete electrophorogram as shown in fig. 1.Wherein snakehead and spot snakehead can adopt usual manner such as form to distinguish, and snakehead and spot snakehead etc. also can adopt usual manner such as form to distinguish.
6.2 the sequencing result size after amplified production reclaims is 201bp, carries out blast(http with the Mitochondrial DNA of snakehead: //blast.ncbi.nlm.nih.gov/) to compare, the segment-similarity that records sequence and Mitochondrial DNA is 99%, specifically sees Fig. 2.Therefore get rid of the possibility that the BY14 primer is influenced by nuclear gene group DNA, should be the amplified reaction that carries out as template with Mitochondrial DNA.
So, the Mitochondrial DNA sequencing result of snakehead, snakehead, black snakehead (snakehead ♀ * snakehead ♂), 4 kinds of fishes of spot snakehead (snakehead ♀ * snakehead ♂) explanation Mitochondrial DNA is strict with matrocliny really among the present invention, and the filial generation Mitochondrial DNA comes from female parent.
Can get rid of the influence of nuclear gene group DNA among the present invention based on the design of primers of mtdna sequence, can deal with problems very easily.
Consider accuracy and terseness among the present invention, this experiment selects for use 30 samples of every kind of fish to verify.PCR through every group of 30 fishes detects, and electrophoresis result shows that 30 fishes of snakehead group and spot snakehead group can both amplify the purpose band, and snakehead group and black snakehead group then do not have band.This presentation of results Auele Specific Primer has the specificity of planting.
Though what authentication method utilized among the present invention is the mtDNA mark, does not need to extract separately mtDNA and identifies.A small amount of mtDNA in the conventional full genome that extracts can utilize primer to pass through pcr amplification, just can access the result behind the electrophoresis.Cycle time is short, and method simply can be carried out in enormous quantities, can obtain the result in one day.
Authentication method can simply be told the difference of two cross-fertilize seed for differentiating that by molecular method the cross-fertilize seed of reciprocal cross provides a kind of method later at the Auele Specific Primer that mtDNA designs among the present invention, and two parents also can use for Rapid identification.
Above-described embodiment is preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included in protection scope of the present invention.
Claims (4)
1. labeled primer of identifying snakehead, snakehead and first familiar generation spot snakehead and black snakehead based on mtdna sequence, it is characterized in that: described labeled primer is BY14, described labeled primer BY14 is made up of left end primer sequence BY14F and right-hand member primer sequence BY14R, wherein said left end primer sequence BY14F is: 5 '-CCCGTCACCCTCACCTAA-3 ', right-hand member primer sequence BY14R is: 5 '-GCGTGGGGCTACCTGTTC-3 '.
2. one kind is utilized the described labeled primer based on mtdna sequence evaluation snakehead, snakehead and first familiar generation spot snakehead and black snakehead of claim 1 to identify that snakehead, snakehead and Eclectics F1 thereof are for the method for spot snakehead and black snakehead, it is characterized in that: utilize the labeled primer in the claim 1, amplification snakehead, snakehead, spot snakehead and black snakehead, wherein snakehead and spot snakehead have the PCR specific band to occur at the 201bp place, snakehead and black snakehead then do not have band to occur, thereby identify spot snakehead and black snakehead.
3. labeled primer evaluation snakehead, snakehead and the Eclectics F1 thereof that identifies snakehead, snakehead and first familiar generation spot snakehead and black snakehead based on mtdna sequence according to claim 2 is characterized in that for the method for spot snakehead and black snakehead described labeled primer obtains by the following method:
(1) extract the complete genome DNA of snakehead, snakehead, spot snakehead and black snakehead, and measure the Mitochondrial DNA complete sequence of snakehead, snakehead, spot snakehead and black snakehead, with reference to allele-specific PCR principle, the design labeled primer;
(2) labeled primer with design carries out pcr amplification, and the electrophoresis result of contrast snakehead, snakehead, spot snakehead and 4 kinds of fishes of black snakehead judges tentatively whether this labeled primer can be utilized;
(3) the pcr amplification product purifying is reclaimed, order-checking is compared sequencing result with snakehead and spot snakehead, judges whether the dna fragmentation that pcr amplification obtains is synthesized as template by mtDNA;
(4) labeled primer is carried out pcr amplification to a plurality of snakeheads, snakehead, spot snakehead and black snakehead sample, electrophoresis then, whether the labeled primer of observing design can normally act in great amount of samples, judge the application feasibility of primer, wherein snakehead group and spot snakehead group has the PCR specific band at about 200bp place, and snakehead group and black snakehead group do not have band to occur, and this labeled primer has specificity, can be as the labeled primer of identifying spot snakehead and black snakehead.
4. according to claim 3ly identify that based on mtdna sequence the labeled primer of snakehead, snakehead and first familiar generation spot snakehead and black snakehead identifies that snakehead, snakehead and Eclectics F1 thereof for the method for spot snakehead and black snakehead, is characterized in that: in the step (4) in a plurality of snakeheads, snakehead, spot snakehead and the black snakehead sample every kind of fish have 30 samples at least.
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| CN201310186877.XA CN103276068B (en) | 2013-05-20 | 2013-05-20 | Marking primer and method for identifying channa argus and channa maculate as well as F1-generation channa argus*channa maculate and channa maculate*channa argus hybridized by channa argus and channa maculate |
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Cited By (6)
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| CN106480226A (en) * | 2016-12-29 | 2017-03-08 | 武汉达邦生物科技有限公司 | Ophicephalus arguss male molecular labeling primer and application |
| CN106498086A (en) * | 2016-12-30 | 2017-03-15 | 青岛农业大学 | Purple scallop and bay scallop and its authentication method in the maternal source of backcross progeny |
| CN107217099A (en) * | 2017-06-28 | 2017-09-29 | 中国水产科学研究院珠江水产研究所 | A kind of SNP marker identified available for snakehead genetic sex and supermale fish and its application |
| CN110551689A (en) * | 2019-08-13 | 2019-12-10 | 中国水产科学研究院珠江水产研究所 | Channa argus brain cell line and construction method and application thereof |
| CN116179657A (en) * | 2022-12-30 | 2023-05-30 | 中国水产科学研究院珠江水产研究所 | Primer combination, microsatellite marker combination, multiplex PCR system, method for identifying snakehead, and application of multiplex PCR system |
| CN116732157A (en) * | 2023-03-28 | 2023-09-12 | 中国海洋大学 | Universal molecular marker for sex and variety identification of snakeheads, macula maculata and hybrid snakeheads |
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2013
- 2013-05-20 CN CN201310186877.XA patent/CN103276068B/en not_active Expired - Fee Related
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106480226A (en) * | 2016-12-29 | 2017-03-08 | 武汉达邦生物科技有限公司 | Ophicephalus arguss male molecular labeling primer and application |
| CN106498086A (en) * | 2016-12-30 | 2017-03-15 | 青岛农业大学 | Purple scallop and bay scallop and its authentication method in the maternal source of backcross progeny |
| CN106498086B (en) * | 2016-12-30 | 2019-10-29 | 青岛农业大学 | Purple scallop and bay scallop and its identification method in backcross progeny female parent source |
| CN107217099A (en) * | 2017-06-28 | 2017-09-29 | 中国水产科学研究院珠江水产研究所 | A kind of SNP marker identified available for snakehead genetic sex and supermale fish and its application |
| CN110551689A (en) * | 2019-08-13 | 2019-12-10 | 中国水产科学研究院珠江水产研究所 | Channa argus brain cell line and construction method and application thereof |
| CN116179657A (en) * | 2022-12-30 | 2023-05-30 | 中国水产科学研究院珠江水产研究所 | Primer combination, microsatellite marker combination, multiplex PCR system, method for identifying snakehead, and application of multiplex PCR system |
| CN116179657B (en) * | 2022-12-30 | 2023-09-05 | 中国水产科学研究院珠江水产研究所 | Primer combination, microsatellite marker combination, multiplex PCR system, method for identifying snakehead, and application of multiplex PCR system |
| CN116732157A (en) * | 2023-03-28 | 2023-09-12 | 中国海洋大学 | Universal molecular marker for sex and variety identification of snakeheads, macula maculata and hybrid snakeheads |
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