CN116732186A - Litopenaeus vannamei temperature-related polymorphism microsatellite marker based on transcriptome sequencing and application - Google Patents
Litopenaeus vannamei temperature-related polymorphism microsatellite marker based on transcriptome sequencing and application Download PDFInfo
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Abstract
The invention discloses a litopenaeus vannamei temperature-related polymorphism microsatellite marker based on transcriptome sequencing and application thereof. The microsatellite markers comprise Lv-LTp008, lv-LTp010, lv-LTp012, lv-LTp013, lv-LTp016, lv-LTp024, lv-LTp031, lv-LTp032, lv-LTp034, lv-LTp035, lv-LTp037, lv-LTp038, lv-LTp039, lv-LTp041, lv-LTp047, lv-LTp048 and Lv-LTp049. The microsatellite marker has strong polymorphism and high heterozygosity, and has advantages in the evaluation of antipathogenic strains and common strains; the primers are stable in amplification and good in repeatability, so that the EST-SSR marker and the amplification primers thereof can be used for population identification of disease-resistant strains of litopenaeus vannamei and the like, can be used for genetic diversity analysis, and can be used for auxiliary breeding of good varieties.
Description
Technical field:
the invention belongs to the technical field of biological molecular markers, and particularly relates to a temperature-related polymorphism microsatellite marker of litopenaeus vannamei based on transcriptome sequencing and application thereof.
Background
Litopenaeus vannamei (Litopenaeus vannamei), also known as Pacific prawn (White pacific shrimp), is commonly known as Penaeus vannamei Boone in China. Belonging to the phylum Arthropoda (Arthropoda), the phylum crustacean (Crustaceae), the class Soft first (Malcostraca), the order Octopus (Decapoda), the family of the Paecilomyces (Penaeidae), the genus Litopenaeus (Litopenaeus). The annual output of the Litopenaeus vannamei in 2020 is 186 ten thousand tons, which accounts for 86 percent of the annual output of the Litopenaeus vannamei in China, and the output of the shrimp larvae is 15000 hundred million, so that the Litopenaeus vannamei culture industry becomes the prop industry for the development of fishery in China. The wild population of the litopenaeus vannamei is distributed in central south america, and the Pacific coastal sea area with water temperature ranging from mexico to chile natural water temperature of more than 20 ℃ throughout the year is typical tropical prawn. The coastal areas from south to north and the sea water and fresh water in a plurality of inland areas in China are cultivated. Therefore, low temperature has become one of the main adverse factors affecting the culture of litopenaeus vannamei. The winter temperature in northern China is generally lower than the temperature required by the survival of Litopenaeus vannamei.
Microsatellite DNA, also known as simple repeats (simplesequence repeat, SSR), is typically a tandem repeat consisting of 1-6 nucleotide repeat units, and in eukaryotic genomes, microsatellite DNA is mostly located in non-coding regions of genes, but also in coding sequences and exons. Microsatellites have the advantages of wide distribution, high polymorphism, co-dominant inheritance and the like, and have been widely used in studies for determining genetic differentiation between populations, estimating population structure and genetic diversity of populations [3]. Microsatellite DNA can be classified into genomic SSRs (genomic simple sequence repeat, gSSRs) and expressed sequence tags SSRs (expressed sequence tags-simple sequence repeat, EST-SSRs) according to its source. Compared with gSSR, EST-SSR has the characteristics of high conservation, convenient and quick polymorphism distribution, good species-to-species universality, large quantity and the like because of being derived from transcriptome data, and has become one of effective molecular marker development methods.
Litopenaeus vannamei is a temperature-changing species, and changes in water temperature can cause effects on cellular activity. Studies have shown that the muscle tissue of Litopenaeus vannamei has obvious effect on acute cold stress. At present, the development of SSR markers of the litopenaeus vannamei based on temperature stress EST (Expressed Sequence Tag) is less.
Disclosure of Invention
The invention aims to solve the problems existing in the prior art and provides a litopenaeus vannamei temperature-related polymorphism microsatellite marker based on transcriptome sequencing and application thereof.
In order to achieve the aim of the invention, the litopenaeus vannamei seedlings constructed by teams are utilized to construct a transcriptome library under low-temperature stress, and the constructed sequencing library is sequenced on an Illumina HiSeq 4000 platform (Illumina, san Diego, USA) of Huada genes limited company (Huada genes, shenzhen). The EST-SSR sequences of the litopenaeus vannamei obtained from the database are used for developing the high polymorphic EST-STR markers of the litopenaeus vannamei and providing polymorphic primers, and the high polymorphic molecular markers are provided for genetic diversity research of the litopenaeus vannamei and auxiliary breeding of good varieties.
According to the invention, 50 pairs of primers are selected for PCR amplification of genomic DNA of litopenaeus vannamei from a low-temperature stress litopenaeus vannamei EST-SSR database according to the results of functional prediction and core region repetition difference, wherein 17 pairs of primers can stably amplify target bands. The STR sample is detected by 3730xl equipment, SSR data is analyzed by Genemapper software, and finally 17 primers are determined to be used for researches such as litopenaeus vannamei germplasm resource identification, genetic diversity analysis, molecular marker assisted breeding and the like.
A first object of the invention is to provide a litopenaeus vannamei EST-STR marker, wherein the EST-STR marker numbers are Lv-LTp008, lv-LTp010, lv-LTp012, lv-LTp013, lv-LTp016, lv-LTp024, lv-LTp031, lv-LTp032, lv-LTp034, lv-LTp035, lv-LTp037, lv-LTp038, lv-LTp039, lv-LTp041, lv-LTp047, lv-LTp048, lv-LTp049;
the nucleotide sequence of the Lv-LTp008 is shown in SEQ ID NO. 1:
the nucleotide sequence of the Lv-LTp010 is shown in SEQ ID NO. 2:
the nucleotide sequence of the Lv-LTp012 is shown in SEQ ID NO. 3:
the nucleotide sequence of the Lv-LTp013 is shown in SEQ ID NO. 4:
the nucleotide sequence of the Lv-LTp016 is shown in SEQ ID NO. 5:
the nucleotide sequence of the Lv-LTp024 is shown in SEQ ID NO. 6:
the nucleotide sequence of the Lv-LTp031 is shown in SEQ ID NO. 7:
the nucleotide sequence of the Lv-LTp032 is shown in SEQ ID NO. 8:
the nucleotide sequence of the Lv-LTp034 is shown in SEQ ID NO. 9:
the nucleotide sequence of the Lv-LTp035 is shown in SEQ ID NO. 10:
the nucleotide sequence of the Lv-LTp037 is shown in SEQ ID NO. 11:
the nucleotide sequence of the Lv-LTp038 is shown in SEQ ID NO. 12:
the nucleotide sequence of the Lv-LTp039 is shown in SEQ ID NO. 13:
the nucleotide sequence of the Lv-LTp041 is shown in SEQ ID NO. 14:
the nucleotide sequence of the Lv-LTp047 is shown in SEQ ID NO. 15:
the nucleotide sequence of the Lv-LTp048 is shown in SEQ ID NO. 16:
the nucleotide sequence of the Lv-LTp049 is shown in SEQ ID NO. 17.
The second object of the present invention is to provide an amplification primer for EST-STR labeling of litopenaeus vannamei, comprising:
for the Lv-LTp008 site:
Lv-LTp008F 5'-CTGTATGTTGTGTATGGTGGCAT-3'; lv-LTp008R 5'-ATTTAACTTGCCTTCCTAATGGC-3'; for the Lv-LTp010 site:
Lv-LTp010F 5'-AGACGTTCGTAGTCGAGTTCTGT-3'; lv-LTp010R 5'-CTTCTCCTTCTCTTTTCCTTTCG-3'; for the Lv-LTp012 site:
Lv-LTp012F:5'-GCAAGATTTATTACAGCAGCACC-3'; lv-LTp012R 5'-TGTTTTTGCGTGAATGGTAATCT-3'; for the Lv-LTp013 site:
Lv-LTp013F 5'-AAGACGAAGGCCAAGTAAGTGAT-3'; lv-LTp013R 5'-CATCTCTTTTTCCTTTTCAGTCC-3'; for the Lv-LTp016 site:
Lv-LTp016F 5'-TTTTACAGGCTCTGTTGACACCT-3'; lv-LTp016R 5'-GATGTTGGCCTTATGAAGAAGAA-3'; for the Lv-LTp024 site:
Lv-LTp024F 5'-AATTCGCTCAAAAATTGACAAAA-3'; lv-LTp024R 5'-AGGAGAAGAGTCAGGCTTAAAGG-3'; for the Lv-LTp031 site:
Lv-LTp031F 5'-GCAACAGAAGCATCATTCAACTA-3'; lv-LTp031R 5'-TGTATATGTGCCATGAAGGAGAA-3'; for the Lv-LTp032 site:
Lv-LTp032F:5’-ACACCGATGTGACTTTGTAGGTT-3’；
Lv-LTp032R 5'-TCAATTTACTCGTTCCAAGTAAGAA-3'; for the Lv-LTp034 site:
Lv-LTp034F:5’-AATCAAGCTCTGGTTTCATTGAC-3’；
Lv-LTp034R:5’-CGTTCACTCCCTGAACTTCATAC-3’；
for the Lv-LTp035 site:
Lv-LTp035F 5'-AGAGAACAAGGAAGACCTGGAGT-3'; lv-LTp035R 5'-AAGACAAACTTACGGTCGTTCAG-3'; for the Lv-LTp037 site:
Lv-LTp037F 5'-GTTGGAGACAACACTGGCATTAT-3'; lv-LTp037R 5'-AACTGGAAGTCCACTGTACAAGC-3'; for the Lv-LTp038 site:
Lv-LTp038F 5'-TGTCCTATGTATAGGCTTTTGTGTG-3'; lv-LTp038R 5'-AAATGCAAGCAAACCACATTTAC-3'; for the Lv-LTp039 site:
Lv-LTp039F:5’-CTTTGAAGGTTGGAATGTTTCTG-3’；Lv-LTp039R:5’-GTCCTCAGAATTTGTGGCAA-3’；
for the Lv-LTp041 site:
Lv-LTp041F:5’-TTACATTTATACACAAGCACGCA-3’；
Lv-LTp041R:5’-ACTGTAATAGTCACCGTGCGAAT-3’；
for the Lv-LTp047 site:
Lv-LTp047F:5’-GATACGATCAGTGTCGTGTGAAG-3’；
Lv-LTp047R:5’-TGACGTAGTTGGAGCTGGTG-3’；
for the Lv-LTp048 site:
Lv-LTp048F:5’-GGATGACTTCCAGTCTTCACAAG-3’；
Lv-LTp048R:5’-ACACATCCATACCTTTCCGTAGA-3’；
for the Lv-LTp049 site:
Lv-LTp049F:5’-TGTTTGCTGTATATATGACTGTATGTCC-3’；
Lv-LTp049R:5’-CCATCCACACAAAAACATACACA-3’。
preferably, the 5' end of the forward primer of the amplification primer is marked with a fluorescent group.
Preferably, the fluorescent group is FAM or HEX.
The third object of the invention is to provide a detection kit of Litopenaeus vannamei EST-SSR markers, which comprises the amplification primer.
The fourth object of the invention is to provide a detection method of EST-STR markers of litopenaeus vannamei, which comprises the following steps:
(1) Extracting genomic DNA of litopenaeus vannamei;
(2) Taking the genome DNA extracted in the step (1) as a template, PCR amplification was performed using the above primer set Lv-LTp008-F/R for the Lv-LTp008 site, primer set Lv-LTp010-F/R for the Lv-LTp010 site, primer set Lv-LTp012-F/R for the Lv-LTp012 site, primer set Lv-LTp013-F/R for the Lv-LTp016 site, primer set Lv-LTp016-F/R for the Lv-LTp016 site, primer set Lv-LTp024-F/R for the Lv-LTp024 site, primer set Lv-LTp031-F/R for the Lv-LTp031 site, primer set Lv-LTp032-F/R for the Lv-LTp032 site, primer set Lv-LTp034-F/R for the Lv-LTp034 site, respectively primer pair Lv-LTp035-F/R for Lv-LTp035 site, primer pair Lv-LTp037-F/R for Lv-LTp037 site, primer pair Lv-LTp038-F/R for Lv-LTp038 site, primer pair Lv-LTp039-F/R for Lv-LTp039 site, primer pair Lv-LTp041-F/R for Lv-LTp041 site, primer pair Lv-LTp047-F/R for Lv-LTp047 site, primer pair Lv-LTp048-F/R for Lv-LTp048 site, PCR, performing PCR amplification on the primer pair Lv-LTp049-F/R of the Lv-LTp049 locus;
(3) Typing the PCR product amplified in the step (2) by using a sequencer;
(4) And (3) carrying out genetic diversity analysis on the typing result obtained in the step (3).
Preferably, the PCR amplification reaction system is 25. Mu.L, and comprises: containing Mg 2+ 2.5. Mu.L of 10mM dNTPs 1. Mu. L, taq enzyme 2U, 10. Mu.M forward primer 1. Mu.L, 10. Mu.M reverse primer 1. Mu. L, DNA template 10ng, the remainder being made up to 25. Mu.L by sterile double distilled water.
Preferably, the PCR amplification is performed by the following reaction procedures: pre-denaturation at 95 ℃ for 3 min; denaturation at 94℃for 30 seconds, annealing at 60℃for 30 seconds, extension at 72℃for 30 seconds, 10 cycles total; denaturation at 94℃for 30 seconds, annealing at 55℃for 30 seconds, extension at 72℃for 30 seconds, a total of 35 cycles, and further extension at 72℃for 5-8 minutes.
The fifth object of the invention is to provide the application of the EST-STR marker, the amplification primer, the detection kit or the detection method in the research of the genetic diversity of the Litopenaeus vannamei population, the gene localization, the construction of genetic linkage map, the germplasm identification, the variety classification or the molecular marker assisted breeding.
The invention develops EST-SSR of litopenaeus vannamei based on transcriptome sequences, and 17 litopenaeus vannamei EST-STR markers with high polymorphism are developed and obtained, and the numbers are Lv-LTp008, lv-LTp010, lv-LTp012, lv-LTp013, lv-LTp016, lv-LTp024, lv-LTp031, lv-LTp032, lv-LTp034, lv-LTp035, lv-LTp037, lv-LTp038, lv-LTp039, lv-LTp041, lv-LTp047, lv-LTp048 and Lv-LTp049 respectively. The EST-STR marker of the litopenaeus vannamei can be used for genetic relationship analysis and molecular marker assisted breeding of the litopenaeus vannamei, and lays a foundation for genetic diversity research of the litopenaeus vannamei and assisted breeding of good varieties.
Drawings
FIG. 1 is a statistical view of the temperature transcriptome repeat SSR of Litopenaeus vannamei
Detailed Description
The invention is further illustrated, but not limited, by the following examples.
The experimental methods in the following examples are all conventional methods or performed according to the kit instructions unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. Primer synthesis and sequencing was performed by biological engineering (Shanghai) Inc.
Example 1
The Litopenaeus vannamei EST-SSR marker primer pair developed based on the transcriptome sequence is obtained through the steps of Litopenaeus vannamei transcriptome sequencing, EST-SSR locus identification, SSR primer design, DNA extraction, SSR primer screening, genetic diversity analysis and the like.
Healthy Litopenaeus vannamei having an average body weight of 7.09.+ -. 3.22g was subjected to gradient temperature treatment (9, 10, 11, 12, 13, 14, 15, 18, 21, 24, 27, 30, 33 ℃ C.) for 2 hours, and after 48 hours, recovered to the control temperature (28 ℃ C.) for 2 hours. The temperature of 13 ℃ is detected to be set as a low-temperature emergency group. RNA-seq experiments were performed with 28℃as control and 13℃as low temperature emergency. And taking 20 samples from each group, respectively taking muscle tissues, and extracting genomic DNA of the litopenaeus vannamei by using a marine animal tissue genomic DNA extraction kit (Tiangen Biochemical technology Co., beijing), wherein the operation steps are strictly carried out according to the specification. The quantification of genomic DNA was performed using a NanoDropTM2000 spectrophotometer and the quality was detected by agarose electrophoresis.
As shown in FIG. 1, there are single to hexa-nucleotides and complex repeat types of SSRs present. The dinucleotide repeats are highest in frequency, 7334 (45.53%), and then trinucleotide and mononucleotide repeats are 4451 (27.53%) and 3284 (20.31%), respectively, and the four, five and six nucleotide repeats are low in frequency, which are 3.77%, 0.98% and 2.07% of the total SSR number, respectively.
From the distribution frequency (Table 1), AG/CT repeat units were the most repeated of all dinucleotides, 3183 (50.08%); next are AC/GT 1850 (29.11%); among them, in AG/CT repeats, six times of SSR were repeated, with 1120 (35.19%). Of all the trinucleotide repeat motifs in SSRs, AGG/CCT, AAT/ATT and AAG/CTT are the most common types, 958 (22.60%), 739 (17.45%) and 519 (12.26%), respectively. Of all AGG/CCT types, five replicates are the most abundant class, 464 (48.49%).
TABLE 1 frequency distribution of repeated SSRs of transcriptome di-and tri-nucleotides of Litopenaeus vannamei
Primer design is carried out according to 29763 Unigene sequences containing SSR sites, a pair of primers is synthesized at one site, single base repeat SSR, complex type SSR, SSR with the length less than 18bp and SSR with PCR products not being between 80 and 200bp are eliminated, repeated functional prediction is eliminated, and 216 pairs of candidate SSR primers are obtained in total. Based on the results of temperature or stress-related functional prediction and core repeat differences, 50 pairs of EST-STR primers (as shown in Table 2) were selected for polymorphism screening.
TABLE 2 EST-STR labeled primer characterization table for Litopenaeus vannamei
Randomly extracting 6 parts of extracted litopenaeus vannamei genome DNA to serve as templates, carrying out polymorphism screening on the primers, carrying out PCR gradient amplification on the genome DNA by adopting 50 pairs of primers in table 2, and a 25 mu L reaction system, wherein the reaction system comprises: containing Mg 2+ 2.5. Mu.L of 10mM dNTPs 1. Mu. L, taq enzyme 2U, 10. Mu.M forward primer 1. Mu.L, 10. Mu.M reverse primer 1. Mu. L, DNA template 10ng, the remainder being made up to 25. Mu.L by sterile double distilled water. The reaction procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 94℃for 30 seconds, annealing at 50℃to 60℃for 30 seconds, extension at 72℃for 30 seconds, 10 cycles total; denaturation at 94℃for 30 seconds, annealing at 55℃for 30 seconds, extension at 72℃for 30 seconds, a total of 35 cycles, and further extension at 72℃for 5-8 minutes.
The PCR amplified products were detected by 2% agarose electrophoresis, which showed that 17 pairs of primers can stably amplify the bands at a specific annealing temperature. Sequencing shows that the 17 pairs of primers can amplify target bands, and the loci are respectively: the sequences and characteristics of Lv-LTp008, lv-LTp010, lv-LTp012, lv-LTp013, lv-LTp016, lv-LTp024, lv-LTp031, lv-LTp032, lv-LTp034, lv-LTp035, lv-LTp037, lv-LTp038, lv-LTp039, lv-LTp041, lv-LTp047, lv-LTp048, lv-LTp049 (shown as SEQ ID NO.1-SEQ ID NO. 17) are shown in Table 3.
TABLE 3 17 pairs SSR primer information for polymorphisms in 44 parts of Litopenaeus vannamei resources
And (3) respectively carrying out fluorescent marking on the 5' ends of the upstream primers of the 17 pairs of primers which are primarily screened by using FAM and HEX, and carrying out PCR amplification by taking the extracted 40 litopenaeus vannamei genomic DNAs as templates. The reaction system is the same as above, and the reaction procedure is the same as above except that the annealing temperature is 60 ℃. PCR amplified products were typed using a 3730XL sequencer and specific values of allele fragments were interpreted using genemap 3.2 software, and expected heterozygosity calculated using Cervus 3.0 software, observed heterozygosity. The analysis result of genetic diversity in the disease-resistant strain shows that: the allele factors of the 17 polymorphic microsatellite markers are between 2 and 10; the effective allele factor is between 1.28 and 4.9; observing the heterozygosity between 0.07 and 0.82; the expected heterozygosity is between 0.22 and 0.83; the shannon index was 0.47 to 1.92 (Table 4).
The 17 EST-STR markers are all proved to have high polymorphism, and can be used for analyzing genetic relationship of litopenaeus vannamei and assisting breeding by molecular markers.
TABLE 4 genetic diversity parameters of 17 EST-SSRs in 40 Litopenaeus vannamei derived from disease-resistant lines
(N: number of samples, na: allele, ne: effective allele, I: shannon information index, ho: observed heterozygosity, he: desired heterozygosity, mean: average).
SEQ ID NO.1
SEQ ID NO.2
SEQ ID NO.3
SEQ ID NO.4
SEQ ID NO.5
SEQ ID NO.6
SEQ ID NO.7
SEQ ID NO.8
SEQ ID NO.9
SEQ ID NO.10
SEQ ID NO.11
SEQ ID NO.12
SEQ ID NO.13
SEQ ID NO.14
SEQ ID NO.15
SEQ ID NO.16
SEQ ID NO.17
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (9)
1. The EST-STR marker for the litopenaeus vannamei is characterized in that the EST-STR markers are numbered as Lv-LTp008, lv-LTp010, lv-LTp012, lv-LTp013, lv-LTp016, lv-LTp024, lv-LTp031, lv-LTp032, lv-LTp034, lv-LTp035, lv-LTp037, lv-LTp038, lv-LTp039, lv-LTp041, lv-LTp047, lv-LTp048 and Lv-LTp049;
the nucleotide sequence of the Lv-LTp008 is shown in SEQ ID NO. 1:
the nucleotide sequence of the Lv-LTp010 is shown in SEQ ID NO. 2:
the nucleotide sequence of the Lv-LTp012 is shown in SEQ ID NO. 3:
the nucleotide sequence of the Lv-LTp013 is shown in SEQ ID NO. 4:
the nucleotide sequence of the Lv-LTp016 is shown in SEQ ID NO. 5:
the nucleotide sequence of the Lv-LTp024 is shown in SEQ ID NO. 6:
the nucleotide sequence of the Lv-LTp031 is shown in SEQ ID NO. 7:
the nucleotide sequence of the Lv-LTp032 is shown in SEQ ID NO. 8:
the nucleotide sequence of the Lv-LTp034 is shown in SEQ ID NO. 9:
the nucleotide sequence of the Lv-LTp035 is shown in SEQ ID NO. 10:
the nucleotide sequence of the Lv-LTp037 is shown in SEQ ID NO. 11:
the nucleotide sequence of the Lv-LTp038 is shown in SEQ ID NO. 12:
the nucleotide sequence of the Lv-LTp039 is shown in SEQ ID NO. 13:
the nucleotide sequence of the Lv-LTp041 is shown in SEQ ID NO. 14:
the nucleotide sequence of the Lv-LTp047 is shown in SEQ ID NO. 15:
the nucleotide sequence of the Lv-LTp048 is shown in SEQ ID NO. 16:
the nucleotide sequence of the Lv-LTp049 is shown in SEQ ID NO. 17.
2. An amplification primer labeled with the litopenaeus vannamei EST-STR as set forth in claim 1, comprising:
for the Lv-LTp008 site:
Lv-LTp008F:5’-CTGTATGTTGTGTATGGTGGCAT-3’；
Lv-LTp008R:5’-ATTTAACTTGCCTTCCTAATGGC-3’；
for the Lv-LTp010 site:
Lv-LTp010F:5’-AGACGTTCGTAGTCGAGTTCTGT-3’；
Lv-LTp010R:5’-CTTCTCCTTCTCTTTTCCTTTCG-3’；
for the Lv-LTp012 site:
Lv-LTp012F:5’-GCAAGATTTATTACAGCAGCACC-3’；
Lv-LTp012R:5’-TGTTTTTGCGTGAATGGTAATCT-3’；
for the Lv-LTp013 site:
Lv-LTp013F:5’-AAGACGAAGGCCAAGTAAGTGAT-3’；
Lv-LTp013R:5’-CATCTCTTTTTCCTTTTCAGTCC-3’；
for the Lv-LTp016 site:
Lv-LTp016F:5’-TTTTACAGGCTCTGTTGACACCT-3’；
Lv-LTp016R:5’-GATGTTGGCCTTATGAAGAAGAA-3’；
for the Lv-LTp024 site:
Lv-LTp024F:5’-AATTCGCTCAAAAATTGACAAAA-3’；
Lv-LTp024R 5'-AGGAGAAGAGTCAGGCTTAAAGG-3'; for the Lv-LTp031 site:
Lv-LTp031F 5'-GCAACAGAAGCATCATTCAACTA-3'; lv-LTp031R 5'-TGTATATGTGCCATGAAGGAGAA-3'; for the Lv-LTp032 site:
Lv-LTp032F 5'-ACACCGATGTGACTTTGTAGGTT-3'; lv-LTp032R 5'-TCAATTTACTCGTTCCAAGTAAGAA-3'; for the Lv-LTp034 site:
Lv-LTp034F:5’-AATCAAGCTCTGGTTTCATTGAC-3’；Lv-LTp034R:5’-CGTTCACTCCCTGAACTTCATAC-3’；
for the Lv-LTp035 site:
Lv-LTp035F 5'-AGAGAACAAGGAAGACCTGGAGT-3'; lv-LTp035R 5'-AAGACAAACTTACGGTCGTTCAG-3'; for the Lv-LTp037 site:
Lv-LTp037F 5'-GTTGGAGACAACACTGGCATTAT-3'; lv-LTp037R 5'-AACTGGAAGTCCACTGTACAAGC-3'; for the Lv-LTp038 site:
Lv-LTp038F 5'-TGTCCTATGTATAGGCTTTTGTGTG-3'; lv-LTp038R 5'-AAATGCAAGCAAACCACATTTAC-3'; for the Lv-LTp039 site:
Lv-LTp039F:5’-CTTTGAAGGTTGGAATGTTTCTG-3’；
Lv-LTp039R:5’-GTCCTCAGAATTTGTGGCAA-3’；
for the Lv-LTp041 site:
Lv-LTp041F:5’-TTACATTTATACACAAGCACGCA-3’；
Lv-LTp041R:5’-ACTGTAATAGTCACCGTGCGAAT-3’；
for the Lv-LTp047 site:
Lv-LTp047F:5’-GATACGATCAGTGTCGTGTGAAG-3’；
Lv-LTp047R:5’-TGACGTAGTTGGAGCTGGTG-3’；
for the Lv-LTp048 site:
Lv-LTp048F:5’-GGATGACTTCCAGTCTTCACAAG-3’；
Lv-LTp048R:5’-ACACATCCATACCTTTCCGTAGA-3’；
for the Lv-LTp049 site:
Lv-LTp049F:5’-TGTTTGCTGTATATATGACTGTATGTCC-3’；
Lv-LTp049R:5’-CCATCCACACAAAAACATACACA-3’。
3. the amplification primer of claim 2, wherein the 5' end of the forward primer of the amplification primer is labeled with a fluorescent group.
4. The amplification primer of claim 3, wherein the fluorescent moiety is FAM or HEX.
5. A detection kit for litopenaeus vannamei EST-SSR markers, which is characterized by comprising the amplification primers of claims 2, 3 and 4.
6. A detection method of Litopenaeus vannamei EST-STR markers comprises the following steps:
(1) Extracting genomic DNA of litopenaeus vannamei;
(2) Taking the genome DNA extracted in the step (1) as a template, PCR amplification was performed using the primer pair Lv-LTp008-F/R for the Lv-LTp008 site, the primer pair Lv-LTp010-F/R for the Lv-LTp010 site, the primer pair Lv-LTp012-F/R for the Lv-LTp012 site, the primer pair Lv-LTp013-F/R for the Lv-LTp013 site, the primer pair Lv-LTp016-F/R for the Lv-LTp016 site, the primer pair Lv-LTp024-F/R for the Lv-LTp024 site, the primer pair Lv-LTp031-F/R for the Lv-LTp031 site, the primer pair Lv-LTp032-F/R for the Lv-LTp032 site, the primer pair Lv-LTp034-F/R for the Lv-LTp034 site, respectively of claim 2, 3 or 4 primer pair Lv-LTp035-F/R for Lv-LTp035 site, primer pair Lv-LTp037-F/R for Lv-LTp037 site, primer pair Lv-LTp038-F/R for Lv-LTp038 site, primer pair Lv-LTp039-F/R for Lv-LTp039 site, primer pair Lv-LTp041-F/R for Lv-LTp041 site, primer pair Lv-LTp047-F/R for Lv-LTp047 site, primer pair Lv-LTp048-F/R for Lv-LTp048 site, PCR, performing PCR amplification on the primer pair Lv-LTp049-F/R of the Lv-LTp049 locus;
(3) Typing the PCR product amplified in the step (2) by using a sequencer;
(4) And (3) carrying out genetic diversity analysis on the typing result obtained in the step (3).
7. The method according to claim 6, wherein the PCR amplification is performed with a reaction system of 25. Mu.L, comprising: containing Mg 2+ 2.5. Mu.L of 10 XPCR buffer, 1. Mu.L of 10mM dNTPs),Taq enzyme 2U, 10. Mu.M forward primer 1. Mu.L, 10. Mu.M reverse primer 1. Mu. L, DNA template 10ng, the remainder was made up to 25. Mu.L with sterile double distilled water.
8. The method according to claim 6, wherein the PCR amplification is performed by the following steps: pre-denaturation at 95 ℃ for 3 min; denaturation at 94℃for 30 seconds, annealing at 60℃for 30 seconds, extension at 72℃for 30 seconds, 10 cycles total; denaturation at 94℃for 30 seconds, annealing at 55℃for 30 seconds, extension at 72℃for 30 seconds, a total of 35 cycles, and further extension at 72℃for 5-8 minutes.
9. Use of the EST-STR marker of claim 1, the amplification primers of claims 2, 3 and 4, the detection kit of claim 5 or the detection method of claims 7 and 8 in the study of genetic diversity of a population of litopenaeus vannamei, gene localization, construction of genetic linkage map, germplasm identification, variety classification or molecular marker assisted breeding.
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