CN101942437A - Specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei and application thereof - Google Patents
Specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei and application thereof Download PDFInfo
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Abstract
The invention discloses specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei. The specific primers are five pairs together, and the nucleotide sequences are shown as SEQIDNO:1-10. The invention also discloses application of the specific primers in screening the microsatellite markers of the EST sequences of the Litopenaeus vannamei, performing diversity analysis of germplasm resources of the Litopenaeus vannamei, performing genetic diversity analysis, identifying families, studying molecular population genetics, constructing genetic maps, positioning important economic traits, studying functional genes, and assisting the molecular genetic breeding or culture of the Litopenaeus vannamei. Compared with the prior art, the specific primers for the microsatellite markers have the characteristics of high efficiency, simpleness, convenience, and quickness in screening required microsatellite markers.
Description
Technical field
The invention belongs to molecular biology dna molecular genetic marker technology, be specifically related to marine economic animal Penaeus Environment of Litopenaeus vannamei Low (
Litopenaeus vannamei) EST(Express Sequence Tag, expressed sequence tag) special primer and the application thereof of microsatellite marker.
Background technology
Environment of Litopenaeus vannamei Low (
Litopenaeus vannamei), be commonly called as Penaeus vannamei (
Penaeus vannamei), belong to Arthropoda (Arthropoda), Crustachia (Crustacea), Decapoda (Decapoda), swimming suborder (Natantia), Penaeidae (Penaeidae), Penaeus (
Penaeus),
Lito-penaeusSubgenus is a wide temperature eurysalinity torrid zone shrimps, is middle the highest shrimp species of single rate of three big cultured prawns (Chinese prawn, tigar prawn, Environment of Litopenaeus vannamei Low) in the world at present, occupies critical role in prawn fishery and aquaculture.
In July, 1988, Environment of Litopenaeus vannamei Low is introduced China by the Institute of Oceanology of the Chinese Academy of Sciences from Hawaii, America, artificial propagation in August, 1992 has obtained preliminary success, obtained the shrimp seedling of short run in 1994 by artificial breeding, the pretty Industry Co.,Ltd in sky, Shenzhen in 1999 cooperates with the U.S. three high Biotech Marine, introduce U.S. SPF Environment of Litopenaeus vannamei Low seed shrimp and raising technology, successfully cultivated SPF Environment of Litopenaeus vannamei Low seedling.So far, Environment of Litopenaeus vannamei Low is extensively cultured in China.
In recent years, and though Environment of Litopenaeus vannamei Low China be general seawater, or low salinity seawater and fresh water in culture, all obtained certain progress.At present, Environment of Litopenaeus vannamei Low has been the absolute predominance kind of China's cultured prawn.2007,1,010,000 tons of China's Environment of Litopenaeus vannamei Low cultured outputs accounted for 80 % of China's prawn culturing output, and in the same year, 1,260,000 tons of China's prawn culturing output account for 37 % of world's prawn culturing ultimate production.But the disease of culturing fast development and producing makes the Environment of Litopenaeus vannamei Low resource decay rapidly, and especially since the nineties, prawn disease was broken out, the Environment of Litopenaeus vannamei Low aquaculture was dropped sharply.The popular disease-resistant strain breeding of prawn that makes of prawn disease becomes the problem that must will solve.At present, domestic fishery genetic improvement is mainly based on colony's seed selection, but the offspring that colony's seed selection obtains, sibship is indeterminate between its individuality, causes losing of inbreeding and genetic diversity in the seed selection process inevitably.Therefore, the strain improvement need be carried out with more accurate selection.These methods are badly in need of finding and a kind ofly can be effectively identified the mark of different familys, thereby know the sibship of breeding population, understand pedigree information, effectively reduce inbreeding, and rationally utilize breeding population efficiently.The present invention utilizes bioinformatics method from the little satellite of Environment of Litopenaeus vannamei Low ESTs database search EST, and screening has polymorphic micro-satellite primers, for utilizing the hereditary feature and the group structure of EST microsatellite molecular marker technical study Environment of Litopenaeus vannamei Low breeding population, realize the Preliminary Applications of molecule marker in the disease-resistant growth breeding of Environment of Litopenaeus vannamei Low, lay the foundation for making up high-density Environment of Litopenaeus vannamei Low genetic linkage maps, QTL location important economical trait and carrying out molecular marking supplementary breeding.
Chinese patent publication number CN1233846C(denomination of invention: the detection technique of Chinese prawn S33 microsatellite marker, December 28 2005 day for announcing) and Chinese patent publication number CN1233845C(denomination of invention: the detection technique of Chinese prawn B683 microsatellite marker, December 28 2005 day for announcing) detection technique of Chinese prawn microsatellite marker is disclosed.
In addition, Chinese patent publication number CN101058831(denomination of invention: the screening method of pacific oyster EST micro-satellite mark, open day on October 24th, 2007) a kind of screening method of pacific oyster EST micro-satellite mark is disclosed, the sequence that comprises the ESTs of the Pacific oyster that utilizes the GenBank announcement, utilize little satellite retrieval software SSRhunter to carry out searching of microsatellite locus, carry out screening and separating for 2 ~ 6 base repeating unit multiplicity greater than little satellite segment of 5, thereby obtain containing little satellite multiple ESTs sequence; Flanking sequence at little satellite tumor-necrosis factor glycoproteins two ends designs primer then, and further detection optimization primer becomes microsatellite marker; At last repeatability, stability and the polymorphism of microsatellite marker are carried out comprehensive evaluation, obtain microsatellite marker.
In recent years, along with the research to Environment of Litopenaeus vannamei Low is constantly goed deep into, now existing more than 160,000 ESTs sequence submitted to, and how excavating effective information from these sequences has become one of emphasis of research.Existing report clones important immunogene from these ESTs sequences, and the foundation of the little satellite of Environment of Litopenaeus vannamei Low EST, will for make up the important growth of highdensity genetic linkage maps, marker assisted selection, location, economic characters such as disease-resistant lay the foundation.A large amount of methods of the microsatellite locus of screening polymorphism from the EST library of Environment of Litopenaeus vannamei Low are not reported so far.
Summary of the invention
The objective of the invention is to fill up the blank of Environment of Litopenaeus vannamei Low EST microsatellite marker screening in the prior art, a kind of special primer of Environment of Litopenaeus vannamei Low EST microsatellite marker is provided.
A kind of special primer of Environment of Litopenaeus vannamei Low EST microsatellite marker, its nucleotide sequence is called after Cz20, Cz117, Fx151, Fx197, Fx4 respectively;
Cz20:F:SEQ ID NO:1 wherein, R:SEQ ID NO:2;
Cz117：F：SEQ?ID?NO:3，R：SEQ?ID?NO:4；
Fx151：F：SEQ?ID?NO:5，R：SEQ?ID?NO:6；
Fx197：F：SEQ?ID?NO:7，R：SEQ?ID?NO:8；
Fx4：F：SEQ?ID?NO:9，R：SEQ?ID?NO:10。
The method of the special primer screening Environment of Litopenaeus vannamei Low EST microsatellite marker of above-mentioned Environment of Litopenaeus vannamei Low EST microsatellite marker, may further comprise the steps: utilize the sequence of Environment of Litopenaeus vannamei Low ESTs in the database to splice and cluster, carry out searching of microsatellite locus then, carry out screening and separating for 2 ~ 6 base repeating unit multiplicity greater than little satellite segment of 5, thereby obtain containing little satellite multiple ESTs sequence; Utilize the special primer of Environment of Litopenaeus vannamei Low EST microsatellite marker to increase, same template is carried out 3 repeat amplification protcols after, with PIC_6.0 software evaluation polymorphism, select PIC〉0.25 be microsatellite marker.
Above-mentioned step of screening the little satellite of Environment of Litopenaeus vannamei Low from Environment of Litopenaeus vannamei Low ESTs sequence is: from NCBI (http://www.ncbi.nlm.nih.gov/) database, select EST, and with
Litopenaeus vannameiFor keyword is searched for, the sequence that search is come out is downloaded by the FASTA form, utilizing in batches then, sequence assembly instrument CAP3 software splices and cluster the ESTs sequence of FASTA form, utilizing little satellite to search software SSRhunter again analyzes one by one to these splicings and the good ESTs of cluster, 2 ~ 6 base number of repeat unit are carried out screening and separating greater than little satellite fragment of 5, obtain original est sequence LV_GL_RA09G18f, LV_HC_RA071F01f, LV_HC_RA074D14f, LV_ES_RA17P12f, LV_ES_RA11M06f, adopt 5.0 pairs of above-mentioned est sequences of primer-design software Primer Primer to carry out design of primers, obtain LV_GL_RA09G18f micro-satellite primers Cz20 respectively, LV_HC_RA071F01f micro-satellite primers Cz117, LV_HC_RA074D14f micro-satellite primers Fx151, LV_ES_RA17P12f micro-satellite primers Fx197, LV_ES_RA11M06f micro-satellite primers Fx4.
The parameter of above-mentioned design primer is:
(1) the primer present position undetermined sequence that do not have;
(2) primer can not freestone heart sequence too closely again can not be too far away, about 10 ~ 60 bp;
(3) primer sequence is made up of 18 ~ 27 Nucleotide, mostly is about 23 bp, and product is between 80 ~ 500 bp;
(4) primer sequence is rich in G/C, and G/C content is generally 40 ~ 60 %, the G/C regular distribution, and the G/C content of upstream and downstream primer can not differ too big;
(5) primer is preferably in 5 ' and 3 ' end and contains a G/C, note avoiding continuous A/T to surpass 5 Nucleotide;
(6) the Tm value of primer should be controlled between 57 ~ 68 ℃, if not all rightly can consider to be controlled at 45 ~ 65 ℃, it is too big that the length of two primers and Tm value should difference, and avoid dimer or hairpin structure between the primer as far as possible;
(7) should be low complementary between the primer or do not have complementary, the higher sequence of 3 ' end similarity, otherwise cause mispairing easily; Primer 3 ' brings out existing continuous base more than 3, as GGG or CCC, the wrong probability that causes is increased.
The application of sample parameter of utilizing the Environment of Litopenaeus vannamei Low micro-satellite primers to carry out pcr amplification is: forward and reverse primer respectively is 0.2 μ mol/L, 0.2 mmol/L dNTP, 1 * PCR Buffer, 2.0 mmol/L Mg
2+, 0.6 U Taq enzyme, 50 ng dna profilings are supplied 20 μ l with distilled water.
The above-mentioned special primer of EST microsatellite marker that utilizes screens in the method for Environment of Litopenaeus vannamei Low EST microsatellite marker, each is respectively the pcr amplification reaction system the primer annealing temperature: SEQ ID NO:1 ~ 2 are 55 ℃, SEQ ID NO:3 ~ 4 are 48 ℃, SEQ ID NO:5 ~ 6 are 55 ℃, SEQ ID NO:7 ~ 8 are 51 ℃, and SEQ ID NO:9 ~ 10 are 50 ℃.
Utilize the method for the special primer detection microsatellite polymorphism of above-mentioned Environment of Litopenaeus vannamei Low EST microsatellite marker, the different Environment of Litopenaeus vannamei Low genomic templates in source is carried out pcr amplification, its product is electrophoresis on 6% polyacrylamide gel, utilize difference in length be the DNA ladder of 50 bp and PBR322 Marker as molecular weight standard, detect the polymorphism of little satellite.
The application of the special primer of above-mentioned Environment of Litopenaeus vannamei Low EST microsatellite marker in the location of Environment of Litopenaeus vannamei Low germ plasm resource diversity analysis, analysis of genetic diversity, family evaluation, molecular population genetics research, construction of genetic atlas, important economical trait, the research of functional gene, auxiliary Environment of Litopenaeus vannamei Low molecular genetic breeding or breed.
The present invention has following beneficial effect:
(1) the present invention is particularly suitable for having the species of a large amount of est sequences, by splicing and the cluster to a large amount of est sequences, searches earlier relatively in the past and carries out cluster analysis science and saving time more behind the microsatellite sequence.
(2) the present invention can be used for genetic marker between Environment of Litopenaeus vannamei Low colony, genealogical identification or construction of genetic atlas etc., and purposes is more extensive.
Description of drawings
Fig. 1 is the UPGMA genealogical tree of 3 Environment of Litopenaeus vannamei Low colonies, and wherein ZX-1 is " in emerging No. 1 " Environment of Litopenaeus vannamei Low, and JK is the Environment of Litopenaeus vannamei Low from the Oceanic Institute of Hawaii import, and Market is the Environment of Litopenaeus vannamei Low that market is bought.
Fig. 2 is the genetic distance UPGMA cluster result of 90 individualities in " in emerging No. 1 " Environment of Litopenaeus vannamei Low, import Environment of Litopenaeus vannamei Low, 3 colonies of market Environment of Litopenaeus vannamei Low, it wherein 1 ~ 30 is " in emerging No. 1 " Environment of Litopenaeus vannamei Low colony, 31 ~ 60 is market Environment of Litopenaeus vannamei Low colony, and 61 ~ 90 are import Environment of Litopenaeus vannamei Low colony.
Embodiment
Embodiment 1: the determining of the screening of microsatellite locus and polymorphism mark thereof
In the experiment used Environment of Litopenaeus vannamei Low from random in the Environment of Litopenaeus vannamei Low of buying on the market, in the anti-WSSV Environment of Litopenaeus vannamei Low of Zhanjiang 863 base seed selections " in emerging No. 1 " family, the Environment of Litopenaeus vannamei Low of Oceanic Institute of Hawaii import.
1, the screening of the source of microsatellite locus and microsatellite sequence
From NCBI (http://www.ncbi.nlm.nih.gov/) database collect and download (with the FASTA form) existing Environment of Litopenaeus vannamei Low (
Litopenaeus vannamei) est sequence, utilizing in batches, sequence assembly instrument CAP3 software splices and cluster the ESTs sequence of FASTA form, utilize microsatellite locus to search software SSRhunter then it is carried out the search of microsatellite sequence, carry out screening and separating for 2 ~ 6 base number of repeat unit greater than little satellite fragment of 5, obtain original est sequence LV_GL_RA09G18f, LV_HC_RA071F01f, LV_HC_RA074D14f, LV_ES_RA17P12f, LV_ES_RA11M06f.
2, the design of primers of micro-satellite primers mark
Utilize Primer Primer 5.0 design primers at the two ends of little satellite tumor-necrosis factor glycoproteins flanking sequence; Design of primers requires:
(1) the primer present position undetermined sequence that do not have;
(2) primer can not freestone heart sequence too closely again can not be too far away, about 10 ~ 60 bp;
(3) primer sequence is made up of 18 ~ 27 Nucleotide, mostly is about 23 bp, and product should be between 80 ~ 500 bp;
(4) primer sequence should be rich in G/C, and G/C content is generally 40 ~ 60%, the G/C regular distribution, and the G/C content of upstream and downstream primer can not differ too big; G/C content is greater than 40%;
(5) primer is preferably in 5 ' and 3 ' end and contains a G/C, note avoiding continuous A/T to surpass 5 Nucleotide;
(6) the Tm value of primer should be controlled between 57 ~ 68 ℃, if not all rightly can consider to be controlled at 45 ~ 65 ℃, it is too big that the length of two primers and Tm value should difference, and avoid dimer or hairpin structure between the primer as far as possible;
(7) should be to hang down complementarity or do not have complementarity between the primer, the higher sequence of 3 ' end similarity, otherwise cause mispairing easily; Primer 3 ' brings out existing continuous base more than 3, as GGG or CCC, the wrong probability that causes is increased.
Adopt 5.0 pairs of above-mentioned est sequences of primer-design software Primer Primer to carry out design of primers, obtain LV_GL_RA09G18f micro-satellite primers Cz20 respectively, LV_HC_RA071F01f micro-satellite primers Cz117, LV_HC_RA074D14f micro-satellite primers Fx151, LV_ES_RA17P12f micro-satellite primers Fx197, LV_ES_RA11M06f micro-satellite primers Fx4.
Table 1 Environment of Litopenaeus vannamei Low micro-satellite primers sequence and feature
3, the screening of micro-satellite primers and optimization
Different primers is optimized according to different Tm values, between the annealing temperature screening area is 48 ~ 66 ℃ 7 thermogrades are set altogether: 48,51,54,57,60,63,66 ℃.The PCR reaction system is 20 μ l, comprises: forward and reverse primer respectively is 0.2 μ mol/L, 0.2 mmol/L dNTP, 1 * PCR Buffer, 2.0 mmol/L Mg
2+, 0.6 U Taq enzyme, 50 ng dna profilings are supplied 20 μ l with distilled water.The PCR response procedures is: 94 ℃ of pre-sex change 5 min; 94 ℃ of sex change 30 s then, gradient (48,51,54,57,60,63,66 ℃) annealing 1 min, 72 ℃ are extended 1 min, repeat this circulation 35 times; Extend 5 min afterwards, 10 ℃ of preservations.Amplified production detects with agarose gel electrophoresis-DNA green coloring system of 1%, filter out do not have assorted band, PCR temperature of reaction that purpose band specificity the is high optimum annealing temperature of little satellite for this reason.
4, microsatellite locus determines
According to the optimum annealing temperature that top step 3 filters out, choose 8 tail Environment of Litopenaeus vannamei Low (field, two tail markets shrimp, two tail import shrimps, the disease-resistant shrimp of two tail familys, the responsive shrimp of two tail familys) and do the detection of microsatellite locus polymorphism.The PCR reaction system is the primer (Cz20, Cz117, Fx151, Fx197, each 0.2 μ M of Fx4) of 20 μ l:0.2 μ mol/L, 0.2 mmol/L dNTP, 1 * PCR Buffer, 2.0 mmol/L Mg
2+, 0.6 U Taq enzyme, 50 ng dna profilings are supplied 20 μ l with distilled water.The PCR response procedures is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30 s then.The optimum annealing temperature that filters out, 1 min anneals.Wherein optimum annealing temperature Cz20 is 55 ℃, and Cz117 is 48 ℃, and FX151 is 55 ℃, and Fx197 is 51 ℃, and Fx4 is 50 ℃.72 ℃ are extended 1 min, repeat this circulation 35 times; Extend 5 min afterwards, 10 ℃ of preservations.Amplified production detects with denaturing polyacrylamide gel electrophoresis-Yin system of dying of 6%: add sex change Buffer 95 ℃ of sex change 5 min afterwards in the PCR product, place cooling on ice afterwards at once, use sample 5 μ l on the microsyringe, the permanent power electrophoresis of 85 W brings to the bottom of offset plate to tetrabromophenol sulfonphthalein, about 120 min, 1 ‰ cma staining, colour developing liquid (30 g NaOH, 0.5 g sodium tetraborate, the formaldehyde of 8 ml, 37 % is dissolved to 2 L ultrapure waters) colour developing, ultrapure water color development stopping, drying are placed on scanning preservation picture in the UTA-2100XL model scanner, with PIC_6.0 software evaluation polymorphism, select PIC〉0.25 be microsatellite marker.
Embodiment 2: application example---the application of microsatellite marker in family is identified
1. extract the Environment of Litopenaeus vannamei Low genome
(1) get the centrifuge tube that Environment of Litopenaeus vannamei Low muscle 30 mg place 2.0 ml, shred, (pH 8.0 for 100 mmol/L NaCl, 10 mmol/L Tris-Cl to add 600 μ l STE lysis buffers; 1 mmol/L EDTA, pH 8.0), 50 μ l SDS and 5 μ l Proteinase Ks (20 mg/ml), mixing;
(2) 56 ℃ of digestion process are up to lysate clarification, centrifugal 3 min of 6000 rpm;
(3) get supernatant, add the saturated phenol of equal-volume (250 μ l), chloroform/primary isoamyl alcohol (24:1) (250 μ l), the mixing number minute of turning upside down gently, albumen, centrifugal 10 min of 10000 rpm are removed in extracting;
(4) get supernatant liquor, repeat one time step 3, till no egg white layer between water and the organic phase;
(5) get supernatant liquor, add equal-volume chloroform/primary isoamyl alcohol (500 μ l), rock 20 min gently, centrifugal 10 minutes of 10000 rpm;
(6) get supernatant liquor, add the NaAc(3 mol/L of 1/10th volumes, pH 5.2), slowly shake up, add the dehydrated alcohol of the precooling of 2 times of volumes, be positioned over about 30 min in-80 ℃ of refrigerators, centrifugal 10 min of 10000 rpm.Nucleic acid is deposited in the pipe end;
(7) abandon supernatant, add 70 % ethanol (1 ml) washing precipitation, 4 ℃ of centrifugal 5 min(8000 rpm);
(8) abandon alcohol, centrifuge tube is placed in the vacuum drier drying precipitated, all volatilize up to ethanol;
(9) add a certain amount of (100 μ l) aseptic ultrapure water or 1 * TE damping fluid and 5 μ l RNaseA(10 mg/ml), 37 ℃ of digestion RNA 30 min ,-20 ℃ of preservations are standby.
2. pcr amplification
The application of sample parameter of pcr amplification: 0.2 μ mol/L primer (Cz20, Cz117, Fx151, Fx197, each 0.2 μ mol/L of Fx4), 0.2 mmol/L dNTP, 1 * PCR Buffer, 2.0 mmol/L Mg
2+, 0.6 U Taq enzyme, 50 ng dna profilings are supplied 20 μ l with distilled water.PCR response procedures: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30 s then, 1 min that anneals, wherein optimum annealing temperature Cz20 is 55 ℃, and Cz117 is 48 ℃, and FX151 is 55 ℃, and Fx197 is 51 ℃, Fx4 is 50 ℃.72 ℃ are extended 1 min, repeat this circulation 35 times; Extend 5 min afterwards, 4 ℃ of preservations.
3. electrophoresis detection
Pcr amplification product detects with denaturing polyacrylamide gel electrophoresis-Yin system of dying of 6%: add after the sex change Buffer 95 ℃ of sex change 5 minutes in the PCR product, place cooling on ice afterwards at once, use sample 5 μ l on the microsyringe, the permanent power electrophoresis of 85 W brings to the bottom of offset plate to tetrabromophenol sulfonphthalein, about 120 min, 1 ‰ cma staining, colour developing liquid (30 g NaOH, 0.5 g sodium tetraborate, the formaldehyde of 8 ml, 37 % is dissolved to 2 L ultrapure waters) colour developing 5min, ultrapure water color development stopping, drying are placed on scanning preservation picture in the UTA-2100XL model scanner.
The gene type of 4 electrophoretic bands
Utilize POPGENE(Version 3.2) calculating population genetic distance, based on different genetic distances, do not have the resulting dendrogram of inclined to one side calculating as shown in Figure 1 by the UPGMA functional analysis of MEGA software: " in emerging No. 1 " is at first with JK(" in emerging No. 1 " parental population) get together, and then be in the same place with the market shrimp, during this has also verified an emerging colony by import shoal of shrimps body through the seed selection of six generations.
5. cluster analysis
For the further effect of this little satellite of checking in family evaluation and sibship, each individual genotype between our these three colonies is calculated genetic distance with popgene32 Version 1.32, utilize MEGA4.0 software to carry out cluster analysis then based on UPGMA, obtain each individual genetic cluster figure between three colonies, as shown in Figure 2: the 30 tail (1-30 of " in emerging No. 1 " colony, black triangle indicates among the figure) closely get together, just get together then with import shrimp and market shrimp.Further significant difference not before import shrimp and the market shrimp seed selection as can be seen, and in parental population and market shrimp significant difference (P＜0.05) is arranged after the emerging seed selection through 6 generations.
Thus can by these 5 molecule markers and with in emerging No. one 30 tail Environment of Litopenaeus vannamei Low be that basic population is set up family identification fingerprint, carry out the foundation that dna fingerprint identification and cluster analysis are used as the family evaluation with the Environment of Litopenaeus vannamei Low in other sources.
Claims (7)
1. the special primer of an Environment of Litopenaeus vannamei Low EST microsatellite marker is characterized in that totally 5 pairs of special primers, and its nucleotide sequence is respectively shown in SEQ ID NO:1 ~ 10.
2. utilize the special primer of the described Environment of Litopenaeus vannamei Low EST microsatellite marker of claim 1, the application in screening Environment of Litopenaeus vannamei Low EST microsatellite marker, Environment of Litopenaeus vannamei Low germ plasm resource diversity analysis, analysis of genetic diversity, family evaluation, molecular population genetics research, genetic map construction, important economical trait location, functional gene research, auxiliary Environment of Litopenaeus vannamei Low molecular genetic breeding or breed.
3. in the application according to the special primer of the described Environment of Litopenaeus vannamei Low EST microsatellite marker of claim 2, the method of screening Environment of Litopenaeus vannamei Low EST microsatellite marker, it is characterized in that may further comprise the steps: utilize that the Environment of Litopenaeus vannamei Low est sequence splices and cluster in the database, carry out searching of microsatellite locus then; Carry out screening and separating for 2 ~ 6 base repeating unit multiplicity greater than little satellite segment of 5, thereby obtain containing little satellite multiple est sequence; Utilize the special primer of Environment of Litopenaeus vannamei Low EST microsatellite marker to increase, same template is carried out 3 repeat amplification protcols after, with PIC_6.0 software evaluation polymorphism, select PIC〉0.25 est sequence is microsatellite marker.
4. according to the special primer of the described Environment of Litopenaeus vannamei Low EST microsatellite marker of claim 1, it is characterized in that the parameter of design of primers is: the sequence of (1) primer present position is determined; (2) primer freestone heart sequence 10 ~ 60 bp; (3) primer sequence is made up of 18 ~ 27 Nucleotide, and product is between 80 ~ 500 bp; (4) primer sequence G/C content is 40 ~ 60%; (5) A/T is no more than 5 Nucleotide continuously; (6) the Tm value of primer is between 45 ~ 68 ℃; (7) be to hang down complementarity or do not have complementarity between the primer, the continuous base of primer 3 ' end is no more than 3.
5. the special primer that utilizes Environment of Litopenaeus vannamei Low EST microsatellite marker according to claim 2, the method of screening Environment of Litopenaeus vannamei Low EST microsatellite marker, it is characterized in that the reaction system of the little satellite of Environment of Litopenaeus vannamei Low being carried out pcr amplification is: forward and reverse primer respectively is 0.2 μ mol/L, 0.2 mmol/L dNTP, 1 * PCR Buffer, 2.0 mmol/L Mg
2+, 0.6 U Taq enzyme, 50 ng dna profilings complement to 20 μ l with distilled water.
6. according to the special primer of the described EST microsatellite marker of claim 5, the pcr amplification reaction of screening Environment of Litopenaeus vannamei Low EST microsatellite marker method, it is characterized in that each is respectively the primer annealing temperature: SEQ ID NO:1 ~ 2 are 55 ℃, SEQ ID NO:3 ~ 4 are 48 ℃, SEQ ID NO:5 ~ 6 are 55 ℃, SEQ ID NO:7 ~ 8 are 51 ℃, and SEQ ID NO:9 ~ 10 are 50 ℃.
7. utilize the method for the special primer detection microsatellite polymorphism of the described Environment of Litopenaeus vannamei Low EST microsatellite marker of claim 1, it is characterized in that the different Environment of Litopenaeus vannamei Low genomic templates in source is carried out pcr amplification, its product is electrophoresis on 6% polyacrylamide gel, utilize difference in length be the DNA ladder of 50 bp and PBR322 Marker as molecular weight standard, detect the polymorphism of little satellite.
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