CN102146460A - Detecting method for Litopenaeus vannamei Boone LvE165 microsatellite DNA marker - Google Patents
Detecting method for Litopenaeus vannamei Boone LvE165 microsatellite DNA marker Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology DNA marking technology and application, in particular to a detecting method for a Litopenaeus vannamei Boone LvE165 microsatellite DNA marker. The invention provides a Litopenaeus vannamei Boone LvE165 microsatellite specific DNA primer, a PCR (Polymerase Chain Reaction) system utilizing the primer and a detecting method for the Litopenaeus vannamei Boone LvE165 microsatellite DNA marker. The method comprises the following steps of: extracting a Litopenaeus vannamei Boone genome and diluting for later use; designing specific primers at both ends of a sequence by utilizing a Litopenaeus vannamei Boone LvE165 microsatellite DNA core sequence; and then carrying out PCR amplification on genome DNA of different groups of Litopenaeus vannamei Boone or individuals in the group by using the primer, analyzing a product and confirming the genotype of each individual so as to obtain a polymorphism genetic variation map. The invention is mainly applied to Litopenaeus vannamei Boone germplasm resource and genetic diversity analysis, molecular population genetics, construction of the genetic map, and the like.
Description
Technical field
The invention belongs to molecular biology dna marker technology and Application Areas, be specifically related to marine economic animal Penaeus Environment of Litopenaeus vannamei Low (
Litopenaeus vannamei) detection method of LvE165 microsatellite DNA mark.
Background technology
Environment of Litopenaeus vannamei Low (
Litopenaeus vannamei) be a kind of wide salt, eurythermic shrimps.This prawn originates in the western hemisphere, and it is coastal from Mexico southwest mainly to be distributed in Central and South America, the pacific rim through Guatemala, El Salvador, Honduras, Nicaragua, Costa Rica, Panama, Colombia, Ecuador to Peru western part.Environment of Litopenaeus vannamei Low is one of main cultured prawn kind in the whole world, also be the topmost cultured prawn kind of China at present, its culture zone is very extensive, comprise that not only north reaches most of coastland in Hainan in the south from Liaoning, also comprise vast fresh water culture district, suitable cultured area accounts for more than 95% of national shrimp culture area.The cultured output of China's Environment of Litopenaeus vannamei Low surpasses 1,000,000 tons (comprising seawater and freshwater aquiculture), accounts for more than 85% of cultured prawn ultimate production at present.
Along with the research work in biology field deepens continuously to Environment of Litopenaeus vannamei Low, also more and more to the demand of Environment of Litopenaeus vannamei Low molecule marker.Genetic improvement or breed of variety are the power that Environment of Litopenaeus vannamei Low is cultured steady progression, many studies show that, the production traitss such as the speed of growth of heritable variation level and biology, resistance against diseases are closely related, therefore, the molecular genetic marker of exploitation Environment of Litopenaeus vannamei Low, detect the Environment of Litopenaeus vannamei Low genetic diversity, study its genetic construction, and then implement molecular marking supplementary breeding, all have crucial meaning for the breeding and the aquaculture of Environment of Litopenaeus vannamei Low.
EST is meant by the clone of picking at random from the cDNA library, carries out 5 ' or the 3 ' terminal sequence of the cDNA that large scale sequencing obtained, and length is generally 150-500 bp.A large amount of in recent years EST data that increase have fast become the important source of SSR, have the EST of 1%-5% to contain SSR approximately.With the little satellite of gSSR(genome) mark compares, and it is more economical to obtain the EST-SSR mark from est sequence, and characteristics are more arranged; Because EST is the expression fragment of functional gene, the microsatellite marker of finding can be directly relevant with functional gene therein, and might be associated with some production traitss, and these characteristics all have very high using value to genetic map construction and marking supplementary breeding; Simultaneously, because gene codominance and conservative property between different plant species, the EST-SSR that develops from a kind of species might be used for other species research of nearly edge simultaneously, thereby provides new approach for comparative genomics, homologous gene clone.Therefore, EST-SSR has been used to make up genetic map, has separated and has identified aspects such as new gene, gene expression difference research, the research of icp gene group and preparation DNA chip.Utilize the EST microsatellite marker, can associate the Environment of Litopenaeus vannamei Low important economical trait with it, reach the purpose of molecular mark, and can further study the Environment of Litopenaeus vannamei Low functional gene.At present also seldom about the research of Environment of Litopenaeus vannamei Low EST microsatellite marker.
Summary of the invention
The objective of the invention is heritable variation collection of illustrative plates for simple and rapid evaluation Environment of Litopenaeus vannamei Low LvE165 genetic marker locus, enrich existing Environment of Litopenaeus vannamei Low microsatellite marker, seek the microsatellite marker that more polymorphism is good, stability is high, a kind of detection method of Environment of Litopenaeus vannamei Low LvE165 microsatellite DNA mark is provided.
The present invention is achieved by the following scheme: the little satellite specific DNA of a kind of Environment of Litopenaeus vannamei Low LvE165 primer is characterized in that its nucleotide sequence is respectively shown in SEQ ID NO:1 and SEQ ID NO:2.
A kind of PCR reaction system of utilizing above-mentioned dna primer is characterized in that described PCR system is: 10 * PCR damping fluid: 2.0 μ l; The SEQ ID NO:1 of 10mM and SEQ ID NO:2 primer each: 0.2-0.5 μ l; 10mM dNTP:0.4-5 μ l; DdH
2O:14.6-15.9 μ l; 5U/ μ l Taq enzyme: 0.2-0.3 μ l; 10ng Environment of Litopenaeus vannamei Low DNA:1-1.5 μ l.
A kind of detection method of Environment of Litopenaeus vannamei Low LvE165 microsatellite DNA mark, it is characterized in that may further comprise the steps: extract Environment of Litopenaeus vannamei Low genome and diluted for use earlier, utilize Environment of Litopenaeus vannamei Low LvE165 microsatellite DNA core sequence (ATT) n again, n=16-19 wherein, in its sequence design specific primers at both ends, its nucleotide sequence is shown in SEQ ID NO:1-2; Use this primer that genomic dna individual in Environment of Litopenaeus vannamei Low different groups or the colony is carried out pcr amplification then, the PCR product is carried out denaturing polyacrylamide gel detect.Analyze after the band silver that utilizes product to occur dyes colour developing, determine the genotype that each is individual, thereby obtain the polymorphism heritable variation collection of illustrative plates (as shown in Figure 1) of Environment of Litopenaeus vannamei Low at LvE165 genetic marker locus.
The Auele Specific Primer nucleotide sequence of LvE165 microsatellite DNA sequence is respectively: normal chain SEQ ID NO:1:5 '-GCTGGTTGTGGACTCTAA-3 ', and minus strand SEQ ID NO:2: 5 '-CCTTGCATTGATCTGTCA-3 ', annealing temperature is 50 ℃.
Its PCR reaction application of sample parameter: forward and reverse primer respectively is 0.2 uM, 0.2 mM dNTP, 1 * PCR Buffer, 1.5-2.5 mM Mg
2+, 0.6 U Taq enzyme, 50-100 ng dna profiling is supplied 20ul with distilled water; Use this primer the time PCR program parameter be set be: 94 ℃ of pre-sex change 2 minutes; Then 94 ℃ of sex change 30-40 seconds, according to different annealing temperature annealing 30 seconds, 72 ℃ were extended 30 seconds-1 minute, repeated 35 circulations of this reaction.
The detection of PCR product: the genomic dna template is carried out pcr amplification, its product electrophoresis on polyacrylamide gel, utilizing difference in length is DNA ladder and the living worker in PBR322 Marker(Shanghai of 50 bp) as molecular weight marker, amplified production detects with 6%-8% denaturing polyacrylamide gel electrophoresis-Yin system of dying, voltage is to add after the sex change Buffer 95 ℃ of sex change 5 minutes in the 8V/cm:PCR product, place cooling on ice afterwards at once, use sample 5ul on the microsyringe, the permanent power electrophoresis of 85 W brings to the bottom of offset plate to bromjophenol blue, about 120 minutes, EB with final concentration 0.5 μ g/ml is ethidium bromide staining 30 minutes, drying is placed on scanning preservation picture in the UTA-2100XL model scanner, analyze polymorphism, determine the genotype that each is individual, and further obtain the polymorphism collection of illustrative plates of LvE165 genetic marker locus.
The present invention is applicable to Environment of Litopenaeus vannamei Low germ plasm resource and analysis of genetic diversity, and family is identified, molecular population genetics, construction of genetic atlas, the location of important economical trait, the research of functional gene is further used for auxiliary Environment of Litopenaeus vannamei Low molecular genetic breeding and breed.
Existing Environment of Litopenaeus vannamei Low microsatellite marker is abundant not enough, sets up the Environment of Litopenaeus vannamei Low genetic linkage maps, physical map, and the QTL location needs the microsatellite marker that more polymorphism is good, stability is high.Compared with prior art, the present invention has the following advantages:
(1) the present invention can identify the heritable variation collection of illustrative plates of Environment of Litopenaeus vannamei Low LvE165 microsatellite genetic marker locus simply and rapidly, and method is easy, and the gained result can detect each individual genotype of Environment of Litopenaeus vannamei Low intuitively.
(2) core of the present invention is the Auele Specific Primer of LvE165 microsatellite DNA mark, its nucleotides sequence is classified as: normal chain 5 '-GCTGGTTGTGGACTCTAA-3 ', minus strand 5 '-CCTTGCATTGATCTGTCA-3 ', annealing temperature is 50 ℃, can present the polymorphism of height heritable variation in the Environment of Litopenaeus vannamei Low population detects.
(3) the present invention is mainly used in Environment of Litopenaeus vannamei Low germ plasm resource and analysis of genetic diversity, and family is identified, molecular population genetics, construction of genetic atlas, the location of important economical trait, the research of functional gene.
Description of drawings
Fig. 1 LvE165 microsatellite DNA mark of the present invention exists, the genotype collection of illustrative plates in the 24 tail Environment of Litopenaeus vannamei Low individualities.
Embodiment
Embodiment 1
1. extract the DNA of Environment of Litopenaeus vannamei Low
The Environment of Litopenaeus vannamei Low material that this experiment is got is 24 DNA samples of random extraction, extracts DNA from refrigerated muscle tissue.Clip 100mg muscle tissue, shred the extraction buffer (6 M Urea(urea) that is placed on 0.7 ml, 10 mM Tris-HCI.125 mM NaCI(sodium-chlor), 1% SDS(dodecyl flesh sodium sulfate), 10 mM EDTA(ethylenediamine tetraacetic acid (EDTA)s), pH=7.5) in, adding final concentration is the Proteinase K (20 mg/ml) of 0.1 mg/ml, and 37 ℃ digest a night.Use phenol: chloroform (1:1) extractive reaction thing three times, extract supernatant liquor with isopyknic chloroform extracting once.Get supernatant liquor and precipitate, be dissolved in then among 500 1 μ l * TE (10 mM Tris.HCI, 1 mM EDTA, pH=8.0) with Virahol.Handled the DNA sample 1 hour with 37 ℃ of RNase (20 μ g/m1), then with the phenol/chloroform extracting solution carry out the extracting of DNA purifying usefulness phenol/chloroform once, the chloroform extracting once.Extract the dehydrated alcohol of supernatant liquor adding two volumes and the 3M NaAc(sodium-acetate of 1/10th times of volumes then), the centrifugal collection of 12000rpm DNA after shaking up, use 70% washing with alcohol twice again, treat that ethanol evaporates fully after, it is stand-by in-20 ℃ of preservations to add 50 μ l, 1 * TE.
2. pcr amplification
The reaction conditions of PCR is: 50ng Environment of Litopenaeus vannamei Low genomic dna, primer SEQ ID NO:1 and SEQ ID NO:2 each 1 μ Μ, 0.2mMdNTP, the Mg2+ of 1 * PCR buffer, 1.5mM, 0.6 UTaq enzyme.The response procedures of PCR is that 94 ℃ of sex change enter circulation after 10 minutes, 94 ℃ of sex change 30 seconds, and 50 ℃ of annealing of Tm 30 seconds, 72 ℃ were extended 1 minute, and 35 circulations are carried out in reaction.Preserve the PCR product for 4 ℃.
3. electrophoresis detection
Pcr amplification product is with 8% polyacrylamide gel electrophoresis, and voltage is 8V/cm, is 0.5 μ g/ml with the EB(final concentration after electrophoresis finishes) dyeed observed and recorded imaging results (as shown in Figure 1) under gel imaging system 30 minutes.
Test-results illustrates that this mark can be used for the detection between the colony, and shows polymorphism, is valuable microsatellite marker.
Embodiment 2
Determining of the screening of microsatellite locus and polymorphic mark thereof
1. the screening of the source of microsatellite locus and microsatellite sequence
From NCBI(http: //www.ncbi.nlm.nih.gov) (with the FASTA form) existing est sequence is collected and downloaded to database, utilize SSRhunter software to search 5736 sequences analyzing gained one by one, 2-6 base repeating unit, multiplicity are separated greater than 5 microsatellite DNA sequence.Adopt the SeqMan among the software DNAstar that the ESTs that contains little satellite is carried out cluster analysis, choose the ESTs design primer of cluster in different contig.
2. microsatellite marker primer design
Utilize the Primerselect among primer-design software Primer Premier 5.0 and the DNAstar to design primer in little satellite repeated flanking sequences; Primer requires to meet the following conditions: (1) primer length is 17-25bp; (2) the GC content requirement is greater than 40%; (3) annealing temperature is greater than 40 ℃; (4) Yu Qi PCR product length is 100-300bp.The primer of design is: normal chain 5 '-GCTGGTTGTGGACTCTAA-3 ', minus strand 5 '-CCTTGCATTGATCTGTCA-3 '.
3. the optimization of primer
Amplified reaction is used MJ-100 and BioRad-200 PCR instrument respectively, and the PCR program is: 94 ℃ of sex change enter circulation after 2 minutes, 94 ℃ of sex change 30 or 40 seconds, and 45 ℃ of annealing 30 seconds, 72 ℃ are extended and did not wait by 1 minute in 30 seconds, and 35 circulations are carried out in reaction.Reaction system is 20 μ l: the Mg that wherein contains forward and reverse primer 0.2 μ Μ, 0.2mMdNTP (mixtures of four kinds of deoxynucleotides), 1 * PCR buffer, 2.0mM
2+, the Taq enzyme, the template amount is respectively 100ng.Template is 5 Environment of Litopenaeus vannamei Low samples at random.The product that obtains of amplification is with polyacrylamide gel electrophoresis-detections of 8%, choose do not have or assortedly be with less, the temperature of PCR correspondence that the specificity product is higher is as the annealing temperature of the best, the Mg of correspondence
2+As best Mg
2+Concentration.
10 * PCR buffer contains 100 mM Tris-HCL (trihydroxy methyl aminomethane-hydrochloric acid pH9.0), 500 mM KCL (Repone K pH9.0), 1% Triton X-100(triton x-100)
4. microsatellite locus determines
According to 45 ℃ of Tm values that screens above and Mg
2+Choose 20 each and every one health check-ups and survey the polymorphism of these microsatellite markers.The response procedures of PCR is that 94 ℃ of sex change enter circulation after 10 minutes, 94 ℃ of sex change 30 seconds, and 45 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, and 35 circulations are carried out in reaction.Reaction system is 20 μ l: the Mg that wherein contains forward and reverse primer 0.2 μ Μ, 0.2mM, 1 * PCR buffer, 1.2mM
2+, the Taq enzyme, the template amount is respectively 50ng.Pcr amplification product is with 8% polyacrylamide gel electrophoresis, voltage is 1-8V/cm, after finishing, electrophoresis uses EB (ethidium bromide, final concentration 0.5 μ g/ml) dyeing is 30 minutes, observed and recorded imaging results under gel imaging system then, analyze and determine polymorphism, filter out 1 site at last as the Environment of Litopenaeus vannamei Low microsatellite marker, the specifying information such as the table one of mark.
Table one. develop the EST-SSR mark of 1 Environment of Litopenaeus vannamei Low (Apostichopus japonicus)
Embodiment 3
1, extracts the DNA of Environment of Litopenaeus vannamei Low
10 Environment of Litopenaeus vannamei Low that present embodiment is got come in 95% alcohol, to soak muscle tissue in extract DNA.Clean twice with ddH2O before extracting, place the extraction buffer (6 M Urea(urea) of 0.7 ml then, 10 mM Tris-HCI.125 mM NaCI(sodium-chlor), 1% SDS(dodecyl flesh sodium sulfate), 10 mM EDTA(ethylenediamine tetraacetic acid (EDTA)s), pH=7.5) in, the adding final concentration is that the Proteinase K (20 mg/ml) of 0.1 mg/ml shreds, and 37 ℃ digest a night.Use phenol: chloroform (1:1) extractive reaction thing three times, extract supernatant liquor with isopyknic chloroform extracting once.Get supernatant liquor and precipitate, be dissolved in then among 500 1 μ l * TE (10 mM Tris.HCI, 1 mM EDTA, pH=8.0) with Virahol.Handled the DNA sample 1 hour with 37 ℃ of RNase (20 μ g/m1), then with the phenol/chloroform extracting solution carry out the extracting of DNA purifying usefulness phenol/chloroform once, the chloroform extracting once.Extract the dehydrated alcohol of supernatant liquor adding two volumes and the 3M NaAc(sodium-acetate of 1/10th times of volumes then), the centrifugal collection of 12000rpm DNA after shaking up, use 70% washing with alcohol twice again, treat that ethanol evaporates fully after, it is stand-by in-20 ℃ of preservations to add 50 μ l, 1 * TE.
2, microsatellite marker primer design
Utilize primer-design software Primer Premier 5.0 design primers in little satellite repeated flanking sequences; Primer requires to meet the following conditions: (1) primer length is 17-25bp; (2) the GC content requirement is greater than 40%; (3) annealing temperature is greater than 40 ℃; (4) Yu Qi PCR product length is 100-300bp.The primer of design is: normal chain 5 '-GCTGGTTGTGGACTCTAA-3 ', minus strand 5 '-CCTTGCATTGATCTGTCA-3 '.
3, the optimization of primer
Amplified reaction is used BioRad-200 PCR instrument respectively, and the PCR program is: 94 ℃ of sex change enter circulation after 2 minutes, 94 ℃ of sex change 40 seconds, and 48 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute, and 35 circulations are carried out in reaction.Reaction system is 25 μ l: the Mg that wherein contains forward and reverse primer 0.2 μ Μ, 0.2mMdNTP (mixtures of four kinds of deoxynucleotides), 1 * PCR buffer, 2.0mM
2+, the Taq enzyme, the template amount is respectively 100ng.The product that obtains of amplification dyes detection with polyacrylamide gel electrophoresis-Yin of 8%, and choosing does not have or assorted band lacks.
4, microsatellite locus is used
According to 48 ℃ of Tm values that screens above and Mg
2+Choose 10 each and every one health check-ups and survey the polymorphism of these microsatellite markers.The response procedures of PCR is that 94 ℃ of sex change enter circulation after 10 minutes, 94 ℃ of sex change 30 seconds, and 45 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, and 35 circulations are carried out in reaction.Reaction system is 20 μ l: the Mg that wherein contains forward and reverse primer 0.2 μ Μ, 0.2mM, 1 * PCR buffer, 1.2mM
2+, the Taq enzyme, the template amount is respectively 50ng.Pcr amplification product is with 8% polyacrylamide gel electrophoresis, and voltage is 8V/cm, and with EB (ethidium bromide, final concentration 0.5 μ g/ml) dyeing 30 minutes, observed and recorded imaging results under gel imaging system was analyzed and determined polymorphism then after electrophoresis finished.
SEQUENCE?LISTING
＜110〉Chinese Academy of Science Nanhai Ocean Research Institute
＜120〉a kind of detection method of Environment of Litopenaeus vannamei Low LvE165 microsatellite DNA mark
<130>
<160> 2
<170> PatentIn?version?3.2
<210> 1
<211> 18
<212> DNA
＜213〉artificial sequence
<400> 1
gctggttgtg?gactctaa 18
<210> 2
<211> 18
<212> DNA
＜213〉artificial sequence
<400> 2
ccttgcattg?atctgtca 18
Claims (7)
1. the little satellite specific DNA of an Environment of Litopenaeus vannamei Low LvE165 primer is characterized in that its nucleotide sequence is respectively shown in SEQ ID NO:1 and SEQ ID NO:2.
2. one kind is utilized the PCR reaction system of dna primer according to claim 1, it is characterized in that described PCR system is: 10 * PCR damping fluid: 2.0 μ l; The SEQ ID NO:1 of 10mM and SEQ ID NO:2 primer each: 0.2-0.5 μ l; 10mM dNTP:0.4-5 μ l; DdH
2O:14.6-15.9 μ l; 5U/ μ l Taq enzyme: 0.2-0.3 μ l; 10ng Environment of Litopenaeus vannamei Low DNA:1-1.5 μ l.
3. the detection method of an Environment of Litopenaeus vannamei Low LvE165 microsatellite DNA mark, it is characterized in that may further comprise the steps: extract Environment of Litopenaeus vannamei Low genome and diluted for use earlier, utilize Environment of Litopenaeus vannamei Low LvE165 microsatellite DNA core sequence (AAT) n again, n=16-19 wherein, in its sequence design specific primers at both ends, its nucleotide sequence is shown in SEQ ID NO:1-2; Use this primer that genomic dna individual in Environment of Litopenaeus vannamei Low different groups or the colony is carried out pcr amplification then, the PCR product is carried out denaturing polyacrylamide gel detect.
4. detection method as claimed in claim 3, it is characterized in that its PCR reaction application of sample parameter is: forward and reverse primer SEQ ID NO:1 and SEQ ID NO:2 respectively are 0.2 uM, 0.2 mM dNTP, 1*PCR damping fluid, 1.5-2.5 mM Mg
2+, 0.6 U Taq enzyme, 50-100 ng dna profiling is supplied 20ul with distilled water; Use this primer the time PCR program parameter be set be: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change 30-40 seconds then, 45-60 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds-1 minute, repeated 35 news rings of this reaction.
5. detection method as claimed in claim 3, it is characterized in that the detection to the PCR product: the genomic dna template is carried out pcr amplification, its product is electrophoresis on 6% ~ 8% polyacrylamide gel, picture is preserved in scanning in dry back, analyze polymorphism, obtain each individual genotype, and further determine the polymorphism heritable variation collection of illustrative plates of LvE165 genetic marker locus.
6. detection method as claimed in claim 5, it is characterized in that utilizing in the detection of PCR product difference in length is that the DNA ladder type of 50 bp and PBR322 mark are as electrophoretic molecular weight marker; Electrophoretic voltage is 1-8V/cm.
7. detection method as claimed in claim 5, it is characterized in that pcr amplification product detects with denaturing polyacrylamide gel electrophoresis-Yin system of dying: add after the sex change damping fluid 95 ℃ of sex change 5 minutes in the PCR product, place cooling on ice afterwards at once, use sample 5ul on the microsyringe, the permanent power electrophoresis of 85 W brings to the bottom of offset plate to bromjophenol blue, after 120 minutes, with the ethidium bromide staining of final concentration 0.5 μ g/ml 30 minutes, observed and recorded imaging results under gel imaging system then.
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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 |
| CN105936937A (en) * | 2016-06-24 | 2016-09-14 | 广东海洋大学 | SNP marker related with low dissolved oxygen survivability of litopenaeus vannamei, screening and applications thereof |
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| CN105936937A (en) * | 2016-06-24 | 2016-09-14 | 广东海洋大学 | SNP marker related with low dissolved oxygen survivability of litopenaeus vannamei, screening and applications thereof |
| CN110343767A (en) * | 2019-06-25 | 2019-10-18 | 天津市水产研究所 | Litopenaeus vannamei microsatellite molecular marker specific primer and its application in analysis of genetic diversity |
| CN110343767B (en) * | 2019-06-25 | 2023-01-13 | 天津市水产研究所 | Specific primer of microsatellite molecular marker of litopenaeus vannamei and application of specific primer in genetic diversity analysis |
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| CN114410806B (en) * | 2021-05-18 | 2023-05-30 | 中国水产科学研究院黄海水产研究所 | Primer combination for microsatellite marker of litopenaeus vannamei and application |
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| CN114854733B (en) * | 2022-04-11 | 2022-10-28 | 广东省农业科学院动物科学研究所 | Microsatellite marker primer combination for constructing DNA fingerprint of litopenaeus vannamei and application and method |
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