CN104611460A - Method for screening and detecting single-nucleotide polymorphic site G642A of marsupenaeus japonicus - Google Patents
Method for screening and detecting single-nucleotide polymorphic site G642A of marsupenaeus japonicus Download PDFInfo
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Abstract
The invention discloses a method for screening and detecting a single-nucleotide polymorphic site G642A of marsupenaeus japonicus, and relates to the marsupenaeus japonicas. A specific primer is designed by a PAMSA method, so that verification and genotyping of a single-nucleotide polymorphic mark of the marsupenaeus japonicas can be conveniently and quickly performed through polyacrylamide gel electrophoresis; the method has the advantages of low screening and detecting expense, high simplicity, convenience and quickness in operation and the like; by the method, parentage determination of the marsupenaeus japonicas, establishment of a genetic linkage map, and other researches are facilitated; under the condition that information of the single-nucleotide polymorphic site of a genome DNA of the marsupenaeus japonicas is unknown, through transcriptome mixed sample sequencing, and by virtue of bioinformatic analysis, the single-nucleotide polymorphic site G642A is screened; the specific primer is designed by the PAMSA method, so that the single-nucleotide polymorphic site G642A of the marsupenaeus japonicas can be conveniently and quickly detected, verified and genotyped through the polyacrylamide gel electrophoresis, the screening and detecting expense is low and the operation is simple, convenient and quick.
Description
Technical field
The present invention relates to Marsupenaeus japonicus, especially relate to a kind of screening and detection method of Marsupenaeus japonicus G642A mononucleotide polymorphism site.
Background technology
Marsupenaeus japonicus (Marsupenaeus japonicus) is distributed in India, West Pacific region, is mainly distributed in coastal waters on the south entrance of Changjiang River in China.It is Fresh & Tender in Texture good to eat, can live transport, and selling price is high, is one of main shrimps in culture of China.Current China is the offspring catching wild close shrimp without the sea of seed selection for the Marsupenaeus japonicus seed cultivated substantially, and the speed of growth, the resistance against diseases of seed are uneven, and raiser is often because fry quality problem causes cultivating unsuccessfully.The breed breeding carrying out Marsupenaeus japonicus is conducive to the sustainable of its industry and develops in a healthy way.Marsupenaeus japonicus is because special spermatheca structure, and spermatophore implantation technique fails effectively to break through so far, and seed selection individuality and family tree need a large amount of DNA molecular marker ability precise Identifications.The people such as Jerry once carried out Marsupenaeus japonicus pedigree qualification work, because adopt microsatellite marker limited amount and the factors such as amorphs cause pedigree identify accuracy rate less than 50% (Moore S S, Whan V, Davis G P, et al.Thedevelopment and application of genetic markers for the Kuruma prawn Penaeusjaponicus [J] .Aquaculture, 1999,173 (1): 19-32).Visible sufficient amount DNA molecular marker is the important prerequisite guarantee of Marsupenaeus japonicus individual recognition, pedigree qualification.But, compared with other cultured prawn kinds, although the report of the existing RAPD DNA marker of current Marsupenaeus japonicus and microsatellite DNA mark exploitation, but the quality and quantity of mark still can not meet the demand (Song Linsheng of the researchs such as pedigree qualification, genetic linkage maps, Xiang Jianhai, Li Chenxi, etc. the RAPD marker research [J] of japonicus wild stocks and cultured populations genetic construction. Oceanologia et Limnologia Sinica, 1999 (3): 261-266; Zhuan Zhimeng, Kong Jie, Shi Tuo, etc. japonicus is wild analyzes [J] with the RAPD of cultured population genetic diversity. and natural science is in progress, and 2001 (3): 28-33; Zhao Y, Li Q.Characterization of expressed sequence tag ?derivedmicrosatellites from the kuruma prawn, Marsupenaeus japonicus [J] .Molecular ecologynotes, 2007,7 (6): 1248-1250).
As the DNA molecular marker of a new generation, single nucleotide polymorphism is the molecule marker that in genome, content is the abundantest, and genetic stability is high, somatotype is accurate, be easy to the detection realizing high-throughput, automatization, be the investigation of current Marsupenaeus japonicus breeding population genetic background, one of first-selected DNA molecular marker that individual seed selection, family tree qualification and high-density genetic linkage maps build.But Marsupenaeus japonicus DNA information is deficient, about its single nucleotide polymorphism exploitation, application research there is not been reported.In Penaeidae, the people such as the people such as Yang Yu and Matthew Baranski report Environment of Litopenaeus vannamei Low (Litopenaeus vannamei) (Yu Y respectively, Wei J, Zhang X, et al.SNP Discovery in theTranscriptome of White Pacific Shrimp Litopenaeus vannamei by Next GenerationSequencing [J] .PloS one, 2014, 9 (1): e87218) and tigar prawn (Penaeus monodon) (BaranskiM, Gopikrishna G, Robinson N A, et al.The Development of a High Density Linkage Mapfor Black Tiger Shrimp (Penaeus monodon) Based on cSNPs [J] .PloS one, 2014, 9 (1): e85413) SNP marker screening, the detection and genotyping method adopted is again sequencing and typing and chip typing respectively, label screening, the cost of checking and application is higher, and due to the difference of prawn kind, the mark that two scholars screen can not directly apply in the correlative study of Marsupenaeus japonicus.
Summary of the invention
The object of the present invention is to provide a kind of screening and detection method of Marsupenaeus japonicus G642A mononucleotide polymorphism site.
The present invention includes following steps:
1) the hepatopancreas total serum IgE of 20 Marsupenaeus japonicus individualities is extracted, biased sample carries out transcript profile order-checking, splicing Marsupenaeus japonicus hepatopancreas is with reference to transcript, comparison location order-checking reads end (reads) to reference to transcript screening mononucleotide pleomorphism site, and screen that comparison massfraction is greater than 40, secondary allelotrope frequency more than 200, secondary gene frequency is candidate sequence higher than the site of 20%;
2) gene order of its candidate comp11169 is:
GATGTCGTATTAATGTCTTTATCCAATTAAAAAATCATCATTTATAATTCTAAGCATCTGACGACTATAATATGTAGGTATAACATTGTACTTATACATGGAGAGTCGAAATCTTGAATCATTTAACCTGTTCCTACAATATCACAAGAGACAAGTAATTGAACCAATCACAATCACAAAAGTAAAGGAAAGAAAGATAGAAAAAAAAAGCAAGCGAAGGAGAAATACATATAAATGTTTCCAACTCTTCCCAACCGCCTTTTAGAAGCGCGCCATTTTTTTTACTTCCTGCGCGCGCATTCGCTGGCGTACAAATTGTTAATCTGGTTGGCGTCGCTCTGCTCCATGTGATCCTTGTCGTAGGCCTCGACGAGAATCACGTTAGGATCCGTGGGCACGATGGTTTCGGCGACGCCCCAGTTCACGGAGAAGGAGTAGGTGCCGTAGTGCATGATGCTCTCGTAGTTGTATCCTTCGCCCACGTACTGCCAGTAGGAGTCCTTGTTGAACTGGGACTCCATGCCAGGCTGCACATTTTCGAAGTGAATAGTGACGTAGTAGTCGCGGTCGTTACGGGTGTGCTCGTGGTAGAAGCCGACAGCGTGCATGAGCTCGTGGATGGCCGTGCCCTTGTAGATGCA
gcCGTTGCTGTCTAGAGAGACCCTCTGCTTGCCTCCAATGGTGCCTACGTATGACCA GCATCCGCTGTCGTTCGTGACGATTTCGATGTAGTTGCCCTGAGTGGTTCTCTCGA CGAAGCGGATGCAGGTGCGGGCGTGGAAGTCGTCCATCGCGGAGAGGATGAGGGAC CTCTGGTAGCTGGTGACAGAACTGCCAAACACATAGGGAACGACTCCACCGGGCCA CAAGTACTGCTCCCCAAGGATGGCAGCGCGTTCCTTCCCTGGTTCCTGTCCTGCAA TACCCTTAATGTCACCCTGGAAGAGGTCAGGGTTGTACATGGCCTGAGCCGCCCTT GGGACAACAGGGGTCGCGCCGGCCACGGCCACGACTGCAAGTAAGGCAACGAGCAA CATGCTGGTGCGAAGATGAAGGAGAAAGAGAGGAAACGTGT, wherein overstriking, underlined base is candidate G642A_SNP site,
3) employing PAMSA (PCR amplification of multiple specific alleles) method design Auele Specific Primer is:
Positive strand primer one: 5 '-TAAAACGTGCCCTTGTAGATGAAG-3 ';
Positive strand primer two: 5 '-AAAACCCCTAAAACGTGCCCTTGTAGATTCAA-3 ';
Negative strand primer: 5 '-TTCGTCGAGAGAACCACT-3 ';
4) extract the genomic dna of Marsupenaeus japonicus muscle of back, diluted for 50ng/ μ L;
5) pcr amplification;
In step 5) in, the PCR reaction system of described pcr amplification is 30 μ L, comprises 50ng/ μ L Marsupenaeus japonicus Genomic DNA solution 1 μ L, 10 × PCR buffer 3 μ L, 2.5mM dNTP Mixs 2.4 μ L, 25mM MgCl
24 μ L, 5U/ μ L Taq DNA polymerase 0.15 μ L, the positive strand primer of 10uM one, positive strand primer two and each 0.5 μ L of negative strand primer, add ddH
2o to 30 μ L; Its PCR response procedures is: 94 DEG C of sex change 10min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 40 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.
6) poly-propionic acid amide detected through gel electrophoresis somatotype: pcr amplification reaction product is carried out 6% denaturing polyacrylamide gels electrophoresis, and use silver dye to carry out detection and genotyping, observe under gel imaging system, if some mononucleotide polymorphism sites can amplify 2 kinds of fragments that difference in length is 8 base sizes in different individualities, then can think that this site is polymorphic site, wherein the individuality of single band is homozygote, the individuality of two bands is heterozygote, can accurately judge individual genotype according to fragment length.
The present invention adopts PAMSA (PCR amplification of multiple specific alleles) method design Auele Specific Primer, convenient, fastly can carry out the checking of Marsupenaeus japonicus single nucleotide polymorphism and gene type by poly-propionic acid amide gel electrophoresis, and there is low, the easy and simple to handle advantage such as fast of selective mechanisms expense, fill up the blank of Marsupenaeus japonicus in the researchs such as single nucleotide polymorphism screening and application, be conducive to the researchs such as the qualification of Marsupenaeus japonicus pedigree, genetic linkage maps structure and carry out.In addition, the present invention under the information not having known Marsupenaeus japonicus genomic dna mononucleotide polymorphism site, can be checked order by transcript profile compound sample, utilizes bioinformatic analysis to screen G642A mononucleotide polymorphism site; And adopt PAMSA method design Auele Specific Primer, not only can complete Marsupenaeus japonicus G642A single nucleotide polymorphism by poly-propionic acid amide gel electrophoresis easily and detect, verify and gene type, and selective mechanisms expense is low, fast easy and simple to handle.
Accompanying drawing explanation
Fig. 1 is the mononucleotide polymorphic site G642A_SNP that provides of the embodiment of the present invention 1, and to the individual detection collection of illustrative plates of Marsupenaeus japonicus 24, (numbering 1 ~ 24 is Marsupenaeus japonicus 24 individualities, AA, AG, GG are the genotype of Different Individual, and 100bp, 150bp are the length of Marker different fragments)
Fig. 2 is that the mononucleotide polymorphic site G642A_SNP that provides of the embodiment of the present invention 2 is to Marsupenaeus japonicus G642A_SNP GG type individuality order-checking peak figure.
Fig. 3 is that the mononucleotide polymorphic site G642A_SNP that provides of the embodiment of the present invention 2 is to Marsupenaeus japonicus G642A_SNP AG type individuality order-checking peak figure.
Fig. 4 is that the mononucleotide polymorphic site G642A_SNP that provides of the embodiment of the present invention 2 is to Marsupenaeus japonicus G642A_SNP AA type individuality order-checking peak figure.
Embodiment
Following examples will the invention will be further described by reference to the accompanying drawings.
First the total serum IgE of the compound sample of Marsupenaeus japonicus hepatopancreas is extracted, transcript profile checks order, splicing Marsupenaeus japonicus hepatopancreas is with reference to transcript, comparison location order-checking reads end (reads) to reference to transcript screening mononucleotide pleomorphism site, and screen that comparison massfraction is greater than 40, secondary allelotrope frequency more than 200, secondary gene frequency is candidate sequence higher than the site of 20%; PAMSA (PCR amplification of multiple specific alleles) method is adopted to design the Auele Specific Primer of candidate sequence; Extract Marsupenaeus japonicus muscle of back genomic dna and diluted for use, the Marsupenaeus japonicus genomic dna of Auele Specific Primer to Different Individual of design is used to carry out pcr amplification, PCR primer is carried out to the denaturing polyacrylamide gel electrophoresis of 6%, and determine the genotype of Different Individual according to electrophoretic sheet segment length difference.
Embodiment 1:
1, utilize bioinformatic analysis, obtain the splicing sequence containing candidate's mononucleotide polymorphic site, detailed process is as follows: the transcript profile order-checking first carrying out the Marsupenaeus japonicus hepatopancreas of biased sample, and use trinity software by default parameters splicing hepatopancreas with reference to transcript, using BWA software to read end (reads) by the transcript profile order-checking of default parameters comparison location arrives with reference to transcript, GATK software screening method comparison massfraction is used to be greater than 40, secondary allelotrope frequency is more than 200, secondary gene frequency is candidate locus higher than the site of 20%, screening comp11169 splices sequence and is used for design of primers, its sequence is:
GATGTCGTATTAATGTCTTTATCCAATTAAAAAATCATCATTTATAATTCTAAGCATCTGACGACTATAATATGTAGGTATAACATTGTACTTATACATGGAGAGTCGAAATCTTGAATCATTTAACCTGTTCCTACAATATCACAAGAGACAAGTAATTGAACCAATCACAATCACAAAAGTAAAGGAAAGAAAGATAGAAAAAAAAAGCAAGCGAAGGAGAAATACATATAAATGTTTCCAACTCTTCCCAACCGCCTTTTAGAAGCGCGCCATTTTTTTTACTTCCTGCGCGCGCATTCGCTGGCGTACAAATTGTTAATCTGGTTGGCGTCGCTCTGCTCCATGTGATCCTTGTCGTAGGCCTCGACGAGAATCACGTTAGGATCCGTGGGCACGATGGTTTCGGCGACGCCCCAGTTCACGGAGAAGGAGTAGGTGCCGTAGTGCATGATGCTCTCGTAGTTGTATCCTTCGCCCACGTACTGCCAGTAGGAGTCCTTGTTGAACTGGGACTCCATGCCAGGCTGCACATTTTCGAAGTGAATAGTGACGTAGTAGTCGCGGTCGTTACGGGTGTGCTCGTGGTAGAAGCCGACAGCGTGCATGAGCTCGTGGATGGCCGTGCCCTTGTAGATGCA
gcCGTTGCTGTCTAGAGAGACCCTCTGCTTGCCTCCAATGGTGCCTACGTATGACCA GCATCCGCTGTCGTTCGTGACGATTTCGATGTAGTTGCCCTGAGTGGTTCTCTCGA CGAAGCGGATGCAGGTGCGGGCGTGGAAGTCGTCCATCGCGGAGAGGATGAGGGAC CTCTGGTAGCTGGTGACAGAACTGCCAAACACATAGGGAACGACTCCACCGGGCCA CAAGTACTGCTCCCCAAGGATGGCAGCGCGTTCCTTCCCTGGTTCCTGTCCTGCAA TACCCTTAATGTCACCCTGGAAGAGGTCAGGGTTGTACATGGCCTGAGCCGCCCTT GGGACAACAGGGGTCGCGCCGGCCACGGCCACGACTGCAAGTAAGGCAACGAGCAA CATGCTGGTGCGAAGATGAAGGAGAAAGAGAGGAAACGTGT, wherein overstriking, underlined base are candidate G642A_SNP site.
2, according to the above-mentioned sequence obtained, the G642A_SNP site primer sequence adopting PAMSA (PCR amplification of multiple specific alleles) method to design is:
Positive strand primer one: 5 '-TAAAACGTGCCCTTGTAGATGAAG-3 ';
Positive strand primer two: 5 '-AAAACCCCTAAAACGTGCCCTTGTAGATTCAA-3 ';
Negative strand primer: 5 '-TTCGTCGAGAGAACCACT-3 '.
3, extract Marsupenaeus japonicus muscle of back genomic dna, be diluted to 50ng/ μ L, PCR reaction system is 30 μ L, comprise 50ng/ μ L Marsupenaeus japonicus Genomic DNA solution 1 μ L, 10 × PCR buffer 3 μ L, 2.5mM dNTP Mixs2.4 μ L, 25mM MgCl
24 μ L, 5U/ μ L Taq archaeal dna polymerase 0.15 μ L, the positive strand primer of 10uM one, positive strand primer two and each 0.5 μ L of negative strand primer, add ddH2O to 30 μ L; PCR response procedures is: 94 DEG C of sex change 10min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 40 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.
4, the PCR primer 10 μ L of the Different Individual that step 3 obtains is got respectively, under the firm power of 70W, carry out 6% denaturing polyacrylamide gels electrophoresis 2h, the colour developing of silver dye, Taking Pictures recording result under gel imaging system, according to the genotype (see Fig. 1) between the different discriminate individuals of PCR primer clip size, the genotype of statistics Different Individual, obtains the mapping genetic variations of Marsupenaeus japonicus mononucleotide polymorphic site G642A_SNP.
Embodiment 2:
1, the checking of G642A_SNP site and somatotype is carried out according to screening comp11169 splicing sequences Design sequencing primer in embodiment 1.
The PCR primer of 2, designed sequencing primer amplification should comprise mononucleotide polymorphic G642A_SNP site, and its primer sequence is:
Positive strand primer three: 5 '-CGTGCCCTTGTAGATGCA-3 ';
Negative strand primer: 5 '-TTCGTCGAGAGAACCACT-3 '.
3, the sequencing primer utilizing step 2 to design, carries out pcr amplification to the individuality of 24 in embodiment 1, and PCR reaction system is 30 μ L, comprise 50ng/ μ L Marsupenaeus japonicus Genomic DNA solution 1 μ L, 10 × PCR buffer 3 μ L, 2.5mMdNTP Mixs 2.4 μ L, 25mM MgCl
24 μ L, 5U/ μ L Taq archaeal dna polymerase 0.15 μ L, the positive strand primer of 10uM and each 0.5 μ L of negative strand primer, add ddH2O to 30 μ L; PCR response procedures is: 94 DEG C of sex change 10min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 40 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.
4, Hua Da genome company is sent to check order the PCR primer of step 3, order-checking peak figure uses novoSNP to carry out gene type (the order-checking peak figure of Fig. 2 ~ 4 signal GG, AG, AA type individuality, G642A_SNP polymorphic site is grey square mark base position), the genotype of each sample is judged according to order-checking peak figure, the result that its genotyping result and embodiment 1 gather propionic acid amide electrophoresis is coincide, and shows that the present invention is accurate and effective to Marsupenaeus japonicus mononucleotide polymorphic G642A_SNP site genotyping result.
Claims (2)
1. the screening of Marsupenaeus japonicus G642A mononucleotide polymorphism site and a detection method, is characterized in that comprising the following steps:
1) the hepatopancreas total serum IgE of 20 Marsupenaeus japonicus individualities is extracted, biased sample carries out transcript profile order-checking, splicing Marsupenaeus japonicus hepatopancreas is with reference to transcript, comparison location order-checking reads end to reference to transcript screening mononucleotide pleomorphism site, and screen that comparison massfraction is greater than 40, secondary allelotrope frequency more than 200, secondary gene frequency is candidate sequence higher than the site of 20%;
2) gene order of its candidate comp11169 is:
GATGTCGTATTAATGTCTTTATCCAATTAAAAAATCATCATTTATAATTCTAAGCATCTGACGACTATAATATGTAGGTATAACATTGTACTTATACATGGAGAGTCGAAATCTTGAATCATTTAACCTGTTCCTACAATATCACAAGAGACAAGTAATTGAACCAATCACAATCACAAAAGTAAAGGAAAGAAAGATAGAAAAAAAAAGCAAGCGAAGGAGAAATACATATAAATGTTTCCAACTCTTCCCAACCGCCTTTTAGAAGCGCGCCATTTTTTTTACTTCCTGCGCGCGCATTCGCTGGCGTACAAATTGTTAATCTGGTTGGCGTCGCTCTGCTCCATGTGATCCTTGTCGTAGGCCTCGACGAGAATCACGTTAGGATCCGTGGGCACGATGGTTTCGGCGACGCCCCAGTTCACGGAGAAGGAGTAGGTGCCGTAGTGCATGATGCTCTCGTAGTTGTATCCTTCGCCCACGTACTGCCAGTAGGAGTCCTTGTTGAACTGGGACTCCATGCCAGGCTGCACATTTTCGAAGTGAATAGTGACGTAGTAGTCGCGGTCGTTACGGGTGTGCTCGTGGTAGAAGCCGACAGCGTGCATGAGCTCGTGGATGGCCGTGCCCTTGTAGATGCA
cCGTTGCTGTCTAGAGAGACCCTCTGCTTGCCTCCAATGGTGCCTACGTATGACCA GCATCCGCTGTCGTTCGTGACGATTTCGATGTAGTTGCCCTGAGTGGTTCTCTCGA CGAAGCGGATGCAGGTGCGGGCGTGGAAGTCGTCCATCGCGGAGAGGATGAGGGAC CTCTGGTAGCTGGTGACAGAACTGCCAAACACATAGGGAACGACTCCACCGGGCCA CAAGTACTGCTCCCCAAGGATGGCAGCGCGTTCCTTCCCTGGTTCCTGTCCTGCAA TACCCTTAATGTCACCCTGGAAGAGGTCAGGGTTGTACATGGCCTGAGCCGCCCTT GGGACAACAGGGGTCGCGCCGGCCACGGCCACGACTGCAAGTAAGGCAACGAGCAA CATGCTGGTGCGAAGATGAAGGAGAAAGAGAGGAAACGTGT, wherein overstriking, underlined base is candidate G642A_SNP site,
3) employing PAMSA method design Auele Specific Primer is:
Positive strand primer one: 5 '-TAAAACGTGCCCTTGTAGATGAAG-3 ';
Positive strand primer two: 5 '-AAAACCCCTAAAACGTGCCCTTGTAGATTCAA-3 ';
Negative strand primer: 5 '-TTCGTCGAGAGAACCACT-3 ';
4) extract the genomic dna of Marsupenaeus japonicus muscle of back, diluted for 50ng/ μ L;
5) pcr amplification;
6) poly-propionic acid amide detected through gel electrophoresis somatotype: pcr amplification reaction product is carried out 6% denaturing polyacrylamide gels electrophoresis, and use silver dye to carry out detection and genotyping, observe under gel imaging system, if some mononucleotide polymorphism sites can amplify 2 kinds of fragments that difference in length is 8 base sizes in different individualities, then can think that this site is polymorphic site, wherein the individuality of single band is homozygote, the individuality of two bands is heterozygote, can accurately judge individual genotype according to fragment length.
2. a kind of screening of Marsupenaeus japonicus G642A mononucleotide polymorphism site and detection method as claimed in claim 1, it is characterized in that in step 5) in, the PCR reaction system of described pcr amplification is 30 μ L, comprise 50ng/ μ L Marsupenaeus japonicus Genomic DNA solution 1 μ L, 10 × PCR buffer 3 μ L, 2.5mM dNTP Mixs 2.4 μ L, 25mM MgCl
24 μ L, 5U/ μ L Taq DNA polymerase 0.15 μ L, the positive strand primer of 10uM one, positive strand primer two and each 0.5 μ L of negative strand primer, add ddH
2o to 30 μ L; Its PCR response procedures is: 94 DEG C of sex change 10min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 40 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.
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| CN108467892A (en) * | 2018-01-05 | 2018-08-31 | 广西壮族自治区海洋研究所 | A kind of screening technique of 2 kinds of morphological variation body candidate sequences of Marsupenaeus japonicus |
| CN108504743A (en) * | 2018-01-05 | 2018-09-07 | 广西壮族自治区海洋研究所 | A kind of screening technique of Penaeus monodon A1426G single nucleotide polymorphism candidate sequences |
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| CN113736891A (en) * | 2021-09-10 | 2021-12-03 | 中国水产科学研究院黄海水产研究所 | Molecular marker G2997 for rapidly identifying low-temperature tolerant variety of penaeus japonicus and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146816A (en) * | 2013-02-05 | 2013-06-12 | 南京大学 | DNA molecular marker method for identification of alien invasive species Spartina alterniflora population |
| CN103146817A (en) * | 2013-02-05 | 2013-06-12 | 南京大学 | SNP marker based typing method for spartina alterniflora population |
| CN104450947A (en) * | 2014-12-29 | 2015-03-25 | 中国水产科学研究院黄海水产研究所 | Turbot S11 mononucleotide polymorphic marker detection method |
-
2015
- 2015-03-05 CN CN201510097807.6A patent/CN104611460B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146816A (en) * | 2013-02-05 | 2013-06-12 | 南京大学 | DNA molecular marker method for identification of alien invasive species Spartina alterniflora population |
| CN103146817A (en) * | 2013-02-05 | 2013-06-12 | 南京大学 | SNP marker based typing method for spartina alterniflora population |
| CN104450947A (en) * | 2014-12-29 | 2015-03-25 | 中国水产科学研究院黄海水产研究所 | Turbot S11 mononucleotide polymorphic marker detection method |
Non-Patent Citations (2)
| Title |
|---|
| 张洪伟 等: "日本沼虾ITS1序列分析及SNPs位点的筛选", 《水生生物学报》 * |
| 李小白 等: "转录组测序(RNA-seq)策略及其数据在分子标记开发上的应用", 《中国细胞生物学学报》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105002267A (en) * | 2015-06-15 | 2015-10-28 | 浙江省海洋开发研究院 | Penaeus japonicus molecule marking method and application |
| CN108424971A (en) * | 2018-01-05 | 2018-08-21 | 广西壮族自治区海洋研究所 | A kind of screening technique of litopenaeus vannamei C239T single nucleotide polymorphism candidate sequences |
| CN108467892A (en) * | 2018-01-05 | 2018-08-31 | 广西壮族自治区海洋研究所 | A kind of screening technique of 2 kinds of morphological variation body candidate sequences of Marsupenaeus japonicus |
| CN108504743A (en) * | 2018-01-05 | 2018-09-07 | 广西壮族自治区海洋研究所 | A kind of screening technique of Penaeus monodon A1426G single nucleotide polymorphism candidate sequences |
| CN112634982A (en) * | 2020-11-23 | 2021-04-09 | 上海欧易生物医学科技有限公司 | Method for screening key genes and key protein sets related to research purposes |
| CN112634982B (en) * | 2020-11-23 | 2023-06-16 | 上海欧易生物医学科技有限公司 | Method for screening key genes and key protein sets related to research purposes |
| CN113337578A (en) * | 2021-06-17 | 2021-09-03 | 集美大学 | Method for efficiently screening positive SNP of aquatic animals based on transcriptome data |
| CN113736891A (en) * | 2021-09-10 | 2021-12-03 | 中国水产科学研究院黄海水产研究所 | Molecular marker G2997 for rapidly identifying low-temperature tolerant variety of penaeus japonicus and application thereof |
| CN113736891B (en) * | 2021-09-10 | 2022-04-22 | 中国水产科学研究院黄海水产研究所 | Molecular marker G2997 for rapidly identifying low-temperature tolerant variety of penaeus japonicus and application thereof |
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