CN106978426B - Amplification primer and method for screening Na, K-ATPase alpha subunit gene and SNP (single nucleotide polymorphism) marker of litopenaeus vannamei - Google Patents
Amplification primer and method for screening Na, K-ATPase alpha subunit gene and SNP (single nucleotide polymorphism) marker of litopenaeus vannamei Download PDFInfo
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Abstract
The invention discloses an amplification primer and an amplification method for screening Na, K-ATPase alpha subunit genes and SNP markers of litopenaeus vannamei. The invention designs 12 pairs of primers aiming at the complete mRNA sequence of the Na, K-ATPase alpha subunit of the litopenaeus vannamei, and obtains the complete gene sequence of the Na, K-ATPase alpha subunit of the litopenaeus vannamei through PCR amplification, sequencing and sequence splicing. Designing primers aiming at the obtained sequence segments, and then carrying out SNP site screening to obtain 78 SNP sites in total. The invention establishes a technical system for developing the SNP marker of the Na, K-ATPase alpha subunit gene of the litopenaeus vannamei, and lays a foundation for further screening the SNP marker related to the salinity stress resistance of the litopenaeus vannamei and developing a molecular marker for paternity test of the litopenaeus vannamei, thereby accelerating the breeding process of the good stress-resistant variety of the litopenaeus vannamei.
Description
The technical field is as follows:
the invention relates to the technical field of aquatic organisms, in particular to an amplification primer and an amplification method for screening Na, K-ATPase alpha subunit genes and SNP markers of litopenaeus vannamei.
Background art:
the Litopenaeus vannamei (Litopenaeus vannamei) is commonly called south American white shrimp, is originally produced in south America, is distributed from the southwest coast of Mexico to the pacific coast of the West of Peru, and is the cultured prawn with the largest yield all over the world. In China, the culture areas of the litopenaeus vannamei are distributed in the coastal seawater culture areas and the inland freshwater culture areas, the culture area is about 25 ten thousand hectares, the annual output exceeds 100 ten thousand tons, the yields of the seawater culture areas and the freshwater culture areas respectively account for 50 percent, and the annual output value is 300 plus 400 million yuan, which is a prop industry for aquaculture in China. The litopenaeus vannamei can survive in both seawater and fresh water, which is closely related to the strong osmotic pressure regulation capability of the litopenaeus vannamei, and the osmotic pressure regulation gene plays an important role in the osmotic pressure regulation process of the litopenaeus vannamei.
Among the osmoregulatory genes, the Na, K-ATPase gene plays a prominent role. Na, K-ATPase is an important membrane protein on which organisms live, and is involved in Na on both sides of cells+、K+The active transport of ions across the membrane plays an important role in maintaining the membrane potential of cells and the osmotic pressure balance inside and outside the cells, providing driving force for the cells to transport inorganic ions and nutrients and discharge metabolites, and the like. The Na, K-ATPase gene is an important osmotic pressure regulating gene of crustaceans, and researches show that the expression quantity of the Na, K-ATPase gene in the tissues of the Litopenaeus vannamei after low salinity stress is obviously increased, so that the Na, K-ATPase gene is a functional gene related to the salinity stress resistance of the Litopenaeus vannamei, is expected to be used for developing a low-salinity-resistant molecular marker of the Litopenaeus vannamei, and the breeding process of a low-salinity-resistant excellent variety of the Litopenaeus vannamei is accelerated.
Internationally, the molecular marker-based molecular marker-assisted breeding technology has become a key technology of contemporary aquatic breeding. A Single Nucleotide Polymorphism (SNP), which is a DNA sequence Polymorphism caused by genomic Single Nucleotide variation (including base transition, transversion, Single base insertion or deletion, etc.), is a latest third generation DNA molecular marker. The method has the advantages of huge number, wide distribution, stable heredity, accurate and simple determination, capability of displaying hidden polymorphism which cannot be detected by other technologies and multiple advantages possibly related to gene functions, and is widely applied to paternity test and molecular marker-assisted breeding. Hitherto, a plurality of SNP markers related to traits have been developed by utilizing functional genes of the litopenaeus vannamei, for example, 4 SNP sites which are remarkably related to the growth traits of the litopenaeus vannamei are found on an alpha Amylase (AMY) gene of the litopenaeus vannamei, 1 SNP site which is related to the cold resistance traits of the litopenaeus vannamei is found on a homologous gene TCP-1-eta gene of a heat shock protein 60(HSP60) family member TCP-1, and 1 SNP site which is remarkably related to the WSSV resistance traits of the litopenaeus vannamei is found on a Catalase (CAT) gene of the litopenaeus vannamei. However, no related report of SNP molecular markers related to the low-salt resistance of litopenaeus vannamei exists.
The invention content is as follows:
the invention aims to provide an amplification primer and an amplification method for screening Na, K-ATPase alpha subunit genes and SNP markers of litopenaeus vannamei. The SNP marker is developed by utilizing the salt tolerance stress-resistance related functional gene of the Litopenaeus vannamei, and a foundation is laid for further screening the SNP marker related to the salt tolerance stress-resistance of the Litopenaeus vannamei and developing a molecular marker for paternity identification of the Litopenaeus vannamei, so that the breeding process of the stress-resistance excellent variety of the Litopenaeus vannamei is accelerated.
In order to realize the purpose, the invention takes the complete mRNA sequence of the Na and K-ATPase alpha subunit of the Litopenaeus vannamei obtained by NCBI as the basis, designs a primer to obtain the whole gene sequence of the gene, screens SNP loci aiming at the whole gene sequence of the Na and K-ATPase alpha subunit, develops SNP markers of the Na and K-ATPase alpha subunit genes of the Litopenaeus vannamei, provides an SNP locus amplification primer, establishes a technical system for developing the SNP markers of the Na and K-ATPase alpha subunit genes of the Litopenaeus vannamei, and lays the foundation for further screening the SNP markers related to the salinity resistance of the Litopenaeus vannamei and developing molecular markers for identifying the Litopenaeus vannamei parents.
The first purpose of the invention is to provide a Na, K-ATPase alpha subunit gene amplification primer for the litopenaeus vannamei, which comprises 12 pairs of amplification primers:
primer pair 1: GLvNK-F1: 5'-CACAGACGGTGAAGAATG-3', respectively;
GLvNK-R1：5’-TGGGACCTTGTGCTCATC-3’；
and (3) primer pair 2: GLvNK-F2: 5'-TGGAACTTGATGAGCACAA-3', respectively;
GLvNK-R2：5’-GGCAATGAAGCAGAGGATA-3’；
and (3) primer pair: GLvNK-F3: 5'-CTCCTGCTGTGGATTGGC-3', respectively;
GLvNK-R3：5’-ATGAGAAGACGCCTGTGA-3’；
and (3) primer pair 4: GLvNK-F4: 5'-GGAGCCCAACAAGGACAA-3', respectively;
GLvNK-R4：5’-GACAGCATTGGTGGAGAA-3’；
and (3) primer pair 5: GLvNK-F5: 5'-TTCTCCACCAATGCTGTC-3', respectively;
GLvNK-R5：5’-GGCTACAATGATACCAATGAG-3’；
and (3) primer pair 6: GLvNK-F6: 5'-TGTTGTGTTCCTCATTGGT-3', respectively;
GLvNK-R6：5’-CAGTCTCGTGGATGGATAC-3’；
and (3) primer pair 7: GLvNK-F7: 5'-CAAGTATCCATCCACGAGA-3', respectively;
GLvNK-R7：5’-AGCCTTTGCTTCAGTGGG-3’；
and (3) primer pair 8: GLvNK-F8: 5'-ATTCCCATCAAGGAGGTT-3', respectively;
GLvNK-R8：5’-GGATGTTAGATGTCAGGGT-3’；
and (3) primer pair 9: GLvNK-F9: 5'-CCTGACATCTAACATCCCTGAG-3', respectively;
GLvNK-R9：5’-GCTTGTCGGTGAATGGGT-3’；
a primer pair 10: GLvNK-F10: 5'-CCTGCCATTTCCCTTGCC-3', respectively;
GLvNK-R10：5’-CGCTCACGGAGACCAAAG-3’；
a primer pair 11: GLvNK-F11: 5'-ATCATGGCTGAGAACGGCTTCC-3', respectively;
GLvNK-R11：5’-TGGAGAACGGAAGAGCAG-3’；
primer pair 12: GLvNK-F12: 5'-TTCCGTTCTCCATCCTTA-3', respectively;
GLvNK-R12：5’-ATGGAGCAGTAGTTGACC-3’。
the second purpose of the invention is to provide an amplification method of the Na, K-ATPase alpha subunit gene of the litopenaeus vannamei, which comprises the following steps: PCR amplification products are respectively obtained by taking genomic DNA of litopenaeus vannamei as a template and the primer pairs GLvNK-F1/GLvNK-R1, GLvNK-F2/GLvNK-R2, GLvNK-F3/GLvNK-R3, GLvNK-F4/GLvNK-R4, GLvNK-F5/GLvNK-R5, GLvNK-F6/GLvNK-R6, GLvNK-F7/GLvNK-R7, GLvNK-F8/GLvNK-R8, GLvNK-F9/GLvNK-R9, GLvNK-F10/GLvNK-R10, GLvNK-F11/GLvNK-R11 and GLvNK-F12/GLNK-R12 as amplification primers, then sequencing the amplified product, and then carrying out sequence splicing to obtain the complete gene sequence of Na, K-ATPase alpha subunit of the litopenaeus vannamei.
The reaction system of the PCR amplification is preferably 25 mu L, and comprises: not containing Mg2+10 × PCR buffer 2.5. mu.L, 25mM MgCl22.0. mu.L, 10mM dNTP 0.5. mu.L, 5U/. mu.L LA Taq enzyme 0.2. mu.L, 10. mu.M forward primer 0.5. mu.L, 10. mu.M reverse primer 0.5. mu. L, DNA template 12.5ng, the remainder being made up to 25. mu.L with sterile water.
The reaction procedure of the PCR amplification is preferably as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 2 minutes for 35 cycles; the extension was continued for 10 minutes at 72 ℃.
The third purpose of the invention is to provide an amplification primer for screening the SNP marker of the Na, K-ATPase alpha subunit gene of the litopenaeus vannamei, which comprises 13 pairs of amplification primers:
a primer pair 13: LvNK001-LvNK 011-F: 5'-TGGAACTTGATGAGCACAA-3', respectively;
LvNK001-LvNK011-R：5’-GGCAATGAAGCAGAGGATA-3’；
primer pair 14: LvNK012-LvNK 023-F: 5'-CTCCTGCTGTGGATTGGC-3', respectively;
LvNK012-LvNK023-R：5’-GACTTTGCCTCCTGATAG-3’；
primer pair 15: LvNK024-LvNK 027-F: 5'-GGCGTCTTCTCCTACTATCA-3', respectively;
LvNK024-LvNK027-R：5’-AAAGCAAAGGGAAAGTGA-3’；
primer pair 16: LvNK028-LvNK 030-F: 5'-GCAGTTGTACCTGGGCATTG-3', respectively;
LvNK028-LvNK030-R：5’-GACAGCATTGGTGGAGAA-3’；
a primer pair 17: LvNK031-LvNK 036-F: 5'-TTCTCCACCAATGCTGTC-3', respectively;
LvNK031-LvNK036-R：5’-GGCTACAATGATACCAATGAG-3’；
a primer pair 18: LvNK037-LvNK 039-F: 5'-TGTTGTGTTCCTCATTGGT-3', respectively;
LvNK037-LvNK039-R：5’-AGGTAGGCATTGTTGAAAG-3’；
primer pair 19: LvNK040-LvNK 042-F: 5'-ATTCCCATCAAGGAGGTT-3', respectively;
LvNK040-LvNK042-R：5’-GGATGTTAGATGTCAGGGT-3’；
and (3) primer pair 20: LvNK043-LvNK 046-F: 5'-CATTGCTTACACCCTGAC-3', respectively;
LvNK043-LvNK046-R：5’-CAGGGAATGAACTTGTAGG-3’；
a primer pair 21: LvNK047-LvNK 052-F: 5'-TATTTAGGTGCCTGCCATTT-3', respectively;
LvNK047-LvNK052-R：5’-AGGACGAAATAAGTGGGT-3’；
primer pair 22: LvNK053-LvNK 055-F: 5'-TCGTTTAGTTTACCCACT-3', respectively;
LvNK053-LvNK055-R：5’-TGTGGCTATGTAGATGGA-3’；
primer pair 23: LvNK056-LvNK 063-F: 5'-TTCAGAAGGGATTTCGTC-3', respectively;
LvNK056-LvNK063-R：5’-GTCCATAGTGATCCTCCA-3’；
a primer pair 24: LvNK064-LvNK 074-F: 5'-ACAACAGGCTCATCTCAA-3', respectively;
LvNK064-LvNK074-R：5’-CTGGTGGACAATGGAGTT-3’；
primer pair 25: LvNK075-LvNK 078-F: 5'-GCCGTAACTCCATTGTCC-3', respectively;
LvNK075-LvNK078-R：5’-TGGAGAACGGAAGAGCAG-3’。
the fourth purpose of the invention is to provide a method for screening the SNP marker of the Na, K-ATPase alpha subunit gene of the litopenaeus vannamei, which comprises the following steps: respectively taking a plurality of Litopenaeus vannamei genomic DNAs from different sources as templates, and respectively taking the primer pairs LvNK001-LvNK011-F/LvNK001-LvNK011-R, LvNK-LvNK 023-F/LvNK012-LvNK023-R, LvNK024-LvNK027-F/LvNK024-LvNK027-R, LvNK-LvNK 030-F/LvNK028-LvNK030-R, LvNK031-LvNK036-F/LvNK 055-LvNK 036-R, LvNK-037-LvNK 039-F/LvNK037-LvNK039-R, LvNK-LvNK 042-F/LvNK040-LvNK 042-5639-043-LvNK 046-F/LvNK043-LvNK 046-35048-LvNK 059-LvNK 0426-F/LvNK 0526-LvNK 053-LvNK 0426-LvNK 053-LvNK, LvNK064-LvNK074-F/LvNK064-LvNK074-R, LvNK075-LvNK078-F/LvNK075-LvNK078-R is used as an amplification primer to carry out PCR amplification to respectively obtain amplification products, then the amplification products are sequenced, sequencing graphs of the amplification products obtained by each pair of primer amplification templates are used as a group to be compared, and SNP sites are screened.
The reaction system of the PCR amplification is preferably 25 mu L, and comprises: not containing Mg2+10 × PCR buffer 2.5. mu.L, 25mM MgCl2mu.L of 2.0. mu.L, 10mM dNTP 0.5. mu.L, 5U/. mu.L of high fidelity PCR enzyme 0.2. mu.L, 10. mu.M forward primer 0.5. mu.L, 10. mu.M reverse primer 0.5. mu. L, DNA template 12.5ng, the remainder being made up to 25. mu.L with sterile water.
The reaction procedure of the PCR amplification is preferably as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 1 minute for 35 cycles; the extension was continued for 10 minutes at 72 ℃.
The Na and K-ATPase alpha subunit gene of the litopenaeus vannamei has 78 SNP sites, which are respectively the nucleotide sequence shown in SEQ ID NO.2, wherein the 672 th base is A or G, the 711 th base is T or C, the 752 nd base is A or G, the 773 rd base is A or C, the 785 th base is T or C, the 796 th base is A or T, the 813 th base is A or T, the 846 nd base is T or C, the 860 rd base is A or G, the 865 th base is T or C, the 902 nd base is T or C, the 813 th base is A or T, the 1142 nd base is T or C, the 1155 th base is T or C, the 1207 th base is A or G, the 1210 th base is A or G, the 1213 th base is A or T, the 1436 th base is A or T, the 1439 th base is T or G, the 1548 th base is T or C, and the A or C, 1547 th base is T or G, 1652 th base is T or C, 1852 nd base is A or G, 1954 th base is A or T, 1980 th base is A or C, 1985 th base is A or G, 2743 th base is A or T, 2753 th base is T or G, 2855 th base is T or C, 3032 th base is T or C, 3034 th base is A or G, 3078 th base is A or G, 3326 th base is A or T, 3368 th base is A or C, 3466 th base is A or T, 3912 th base is T or C, 4029 th base is T or C, 4041 th base is T or C, 4548 th base is A or G, 4677 th base is T or C, 4754 th base is A or T, 5221 th base is A or T, 5241 th base is T or C, A5284 th base is T or T, 5684 th base is A or T, 5650 th base is A or T, 5641 th base, 8454 th base is A or G, 8507 th base is A or G, 8520 th base is A or T, 8559 th base is T or G, 8593 th base is A or G, 8796 th base is T or G, 8827 th base is A or T, 8838 th base is T or C, 10799 th base is T or G, 10903 th base is T or C, 10942 th base is A or T, 10965 th base is A or C, 11008 th base is T or C, 11030 th base is T or G, 11057 th base is A or C, 11217 th base is A or T, 11267 th base is T or C, 11272 th base is A or G, 11289 th base is T or G, 11290 th base is T or C, 11298 th base is T or C, 11299 th base is A or T, 11300 th base is T or C, 11364 th base is T or G, 11312 th base is T or G, 11375 th base is T or C, 11331 th base is T or C, 1139 th base, The 11388 th base is T or C, the 11649 th base is A or G, the 11663 th base is A or T, the 11678 th base is T or C, and the 11689 th base is A or T.
The invention obtains the complete mRNA sequence (GenBank: KF765670.1) of the Na and K-ATPase alpha subunit of the litopenaeus vannamei from NCBI, designs 12 pairs of primers based on the sequence, and obtains the complete gene sequence of the Na and K-ATPase alpha subunit of the litopenaeus vannamei with the full length of 12452bp through PCR amplification, sequencing and sequence splicing. Primers are designed aiming at the obtained whole gene sequence segmentation of Na, K-ATPase alpha subunit, PCR amplification is carried out by adopting genomic DNA templates of Litopenaeus vannamei from different sources, SNP locus screening is carried out by a sequencing method, and 78 SNP loci are obtained in total. The invention establishes a technical system for developing the SNP marker of the Na, K-ATPase alpha subunit gene of the litopenaeus vannamei, and lays a foundation for further screening the SNP marker related to the salinity stress resistance of the litopenaeus vannamei and developing a molecular marker for paternity test of the litopenaeus vannamei, thereby accelerating the breeding process of the good stress-resistant variety of the litopenaeus vannamei.
Description of the drawings:
FIG. 1 is a diagram of the structure of the Na, K-ATPase alpha subunit gene of a Litopenaeus vannamei; wherein the numbers represent the number of nucleotides, the light grey represents the exon, and the dark grey represents the intron.
The specific implementation mode is as follows:
the present invention will be further illustrated with reference to the following examples, but is not limited thereto.
The experimental procedures in the following examples were carried out in a conventional manner or according to the kit instructions unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The cloning sequencing and primer synthesis work was done by Shanghai bioengineering, Inc.
1. Obtaining of complete gene sequence of Na, K-ATPase alpha subunit of Litopenaeus vannamei
1.1 extraction of genomic DNA of Litopenaeus vannamei
Taking muscle tissues of the litopenaeus vannamei, extracting the genomic DNA of the litopenaeus vannamei by adopting a marine animal tissue genomic DNA extraction kit (Tiangen Biochemical technology Co., Ltd., Beijing), and strictly performing the operation steps according to the instruction. Genomic DNA quantification was performed using a NanoDropTM2000 spectrophotometer, and quality was checked by agarose electrophoresis.
1.2PCR primer design
A primer is designed to obtain the whole gene sequence of Na, K-ATPase alpha subunit based on the complete mRNA sequence (GenBank: KF765670.1) of the Na, K-ATPase alpha subunit of the Litopenaeus vannamei obtained from NCBI (http:// blast. NCBI. nlm. nih. gov/blast. cgi). The requirements for primer design are as follows: the length of the primer is 18-22bp, the GC content is 40-60%, the Tm value is 50-62 ℃, the difference between the Tm values of the upstream primer and the downstream primer is not more than 5, and primer dimer, hairpin structure, mismatching and the like are avoided as much as possible. 12 pairs of PCR primers were designed and synthesized, and the primer sequences are shown in Table 1:
TABLE 1 characteristics of primers and amplification products for acquisition of Na, K-ATPase alpha subunit whole gene sequence of Litopenaeus vannamei
Note: the numbers in parentheses indicate the nucleotide positions of the primer sequences corresponding to the Na, K-ATPase alpha subunit mRNA sequence (GenBank: KF765670.1) of Litopenaeus vannamei
1.3PCR amplification, sequencing and sequence splicing
Taking the extracted genomic DNA of Litopenaeus vannamei as a template, respectively adopting 12 pairs of primers designed in Table 1 to perform PCR amplification on the DNA, wherein the reaction system is 25 μ L and comprises 10 × PCR buffer (without Mg)2+)2.5μL、MgCl22.0. mu.L (25mM), 0.5. mu.L dNTP (10mM), 0.2. mu.L LA Taq enzyme (5U/. mu.L), 0.5. mu.L forward primer (10. mu.M), 0.5. mu. L, DNA template (25 ng/. mu.L) reverse primer (10. mu.M), and 18.3. mu.L sterile water, the reaction procedure was pre-denaturation at 95 ℃ for 3 minutes, denaturation at 95 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 2 minutes, and re-extension at 72 ℃ for 10 minutes, the PCR amplification products were examined by 1% agarose electrophoresis, showing that the 12 pairs of primers were all able to stably amplify a single band, clone sequencing of the amplification products respectively, showing that 4 pairs of primers amplify the nucleotide sequence of interest, that the amplification products of 8 pairs of primers contain intron sequences in addition to the nucleotide sequence of interest, carrying out sequencing to obtain the full-length spliced nucleotide sequence of shrimp 12452bp, Na-containing complete exon sequences, and 10 nucleotide sequences of 10 cDNA (shown in FIG. 1. 10).
2. Screening of whole gene sequence SNP locus of Na, K-ATPase alpha subunit of litopenaeus vannamei
2.1 extraction of genomic DNA of Litopenaeus vannamei from different sources
Selecting 24 litopenaeus vannamei tails from 24 farms such as Guangdong Shenzhen, Zhuhai, Zhanjiang, Xuwen and Maoyang, respectively taking muscle tissues, extracting genomic DNA of the litopenaeus vannamei by adopting a marine animal tissue genomic DNA extraction kit (Tiangen Biochemical technology Co., Ltd., Beijing), and strictly performing the operation steps according to the instruction. Genomic DNA quantification was performed using a NanoDropTM2000 spectrophotometer, and quality was checked by agarose electrophoresis.
2.2PCR primer design
Primers are designed by segments on the basis of the complete gene sequence (the nucleotide sequence is shown as SEQ ID NO. 1) of the Na, K-ATPase alpha subunit of the Litopenaeus vannamei obtained in the research. The requirements for primer design are as follows: the length of the primer is 18-22bp, the GC content is 40-60%, the Tm value is 50-62 ℃, the difference between the Tm values of the upstream primer and the downstream primer is not more than 5, and primer dimer, hairpin structure, mismatching and the like are avoided as much as possible. A total of 25 PCR primers were designed and synthesized, with the primer sequences shown in table 2:
TABLE 2 primers for screening of Na, K-ATPase alpha subunit whole gene sequence SNP locus of Litopenaeus vannamei and amplification product characteristics
Note: the numbers in parentheses indicate the nucleotide positions of the primer sequences corresponding to the Na, K-ATPase alpha subunit whole gene sequences of the litopenaeus vannamei (the nucleotide sequences are shown as SEQ ID NO. 1)
2.3 screening of SNP loci and genotype analysis of SNP loci
And (3) respectively carrying out PCR amplification by adopting 25 pairs of primers in the table 2 by taking the 24 genomic DNAs of the litopenaeus vannamei extracted in the step 2.1 as templates. The reaction system and reaction procedure are essentially identical to those described in step 1.3, except that: (ii) high fidelity PCR enzyme in PCR System for screening SNP sites
HS DNA Polymerase) replaced LA Taq enzyme, and extension was reduced from 2 min to 1 min at 72 ℃. The PCR amplification products were first detected by 2% agarose electrophoresis and then sequenced using a 3730XL sequencer. Will each beSequencing charts of amplification products obtained by amplifying 24 samples with primers are compared as a group to screen SNP sites, SNP sites are found in the amplification products of 15 pairs of primers (see Table 2), 78 SNP sites are screened in total, the 78 SNP sites are in a sequence shown in SEQ ID NO.2 (the bold part is an intron sequence; the SNP sites are underlined mixed bases, wherein R represents A or G, Y represents C or T, M represents A or C, K represents G or T, S represents C or G, and W represents A or T), and the genotypes and the genotype frequencies of the SNP sites are shown in Table 3.
TABLE 3SNP sites and genotype characteristics in the Na, K-ATPase alpha subunit complete Gene sequence of Litopenaeus vannamei
Sequence listing
<110> Nanhai ocean institute of Chinese academy of sciences
Amplification primer and method for screening Na, K-ATPase alpha subunit gene and SNP (single nucleotide polymorphism) marker of litopenaeus vannamei
<160>4
<210>1
<211>12452
<212>DNA
<213> Litopenaeus vannamei (Litopenaeus vannamei)
<400>1
gtcgtgaagc gcgcgtcgtc gagatcggac gtgccgctgt gccctttagt gtcgaccaac 60
aaacacttca cagacggtga agaatgtgct tgtagcgaaa acctacacag gtcctagagg 120
gcaacattaa gggagtgaac gagtgtacaa agtgaagcct agtggcttgt acataagtgt 180
gtatatacag tgattgttgg tgctggtttg agagccagcg cgtcgccgtt gttcgtggat 240
actgtgtatc ctctgctgcc gtcctcctgc tgagcgccga gtgtcgcctc ttcagcagcc 300
atggccgata cgggcagaac agattcgtat cgacacgcca cggatcgtac catccccgac 360
gataaccgaa cagtgaaagg ggatcctaag tctaagaaaa agccccagaa ggctaaaggg 420
aagaagggag ataaggattt gaatgatctg aagcaggagt tggaacttga tgagcacaag 480
gtcccaattg aggaactctt tcaacgtctc actgttaacc cagacacagt aggtttaccc 540
gagttctttg tagtatcctt ggtatattgc ctaaattaag agtattggta agatttaatc 600
caatagcgac agggaaggaa ttttgggaaa gtgggttaag gagaagtgta cattagcgtg 660
cacagctctg cgcattgggt gattctccag ctattttttg taattacttg ttatgattta 720
atctaaaatc acctgtcgct aataggttag aatagatctt taagagtaga tacacgcaga 780
taaccctcaa aaataaatca aatgtaattc ttacttgtgt actttaatta caaggtgttt 840
ttctgccttg aacatgacta accactccta taaccttgca gggtctatca caaagtgagg 900
ctaagcgccg tattgaacga gatgggccga atgctcttac cccacccaag cagactccag 960
aatgggtcaa gttctgcaaa aacctcttcg gtggtttctc actcctgctg tggattggcg 1020
ctatcctctg cttcattgcc tactcaattg agacagctgc agaagaggag cccaacaagg 1080
acaatgtaag ttttcagtgt tttgttggaa ataagagtat tctttatcta aagagatgat 1140
ttggtaaatg ttaatatgtt gaaaaatatg tacaactaag ggattcaagt gatttgcaat 1200
tttaaagttg aattgtagtt atttaatgtt tttcttttga ttaaggtagt tcatgagaaa 1260
tgtcatactt tgttttaggc caattgtttg aaaatgcatt aggcatagca atatatacat 1320
aatcatatct ataagggcat ttgcatttaa caaacttttt cttccttaag ggtttaattt 1380
gacttgcagc tggtatgcag atgttggagt taaaacattt taatggaccg tgttttgtgt 1440
atttcaatat ccaattcttt ttattagata atgaaactgc tcccatagtt aggtttagag 1500
agaaaaatgt tttattaata gatgtgagga agcataaagt ttaaagttgt gcaggttaag 1560
aattttagag aatataaatc ataataaatg aaagctagat atttctgagt gttagtgtgt 1620
ctcaaccaat ttatcaaagt tcgtaacatc ttatggtgat attttacagt tatacctagg 1680
tgttgtgctg actggtgtgg tgattataac aggcgtcttc tcctactatc aggaggcaaa 1740
gtcctctgct attatggatt catttaagaa catggtgcct caggtaaacc cttagtaatg 1800
ctacatgcct tagttagcta gatgttggtt ctcctctact gttctgatga ggaggaggaa 1860
ggtcctctga tgtgtagtgg gtaggtatgg gatcaatcat ttgaggtaag gttagagaat 1920
tagaccctgg gggagaaact aaacacggat gacatcagag gcttcaaagt cagtgagctc 1980
ataggtagat ggcacctttt ttttttttct tcttcttctt cttcttcctc catgtgaggt 2040
aaggtcatag gaaggaatga tggtctggag tttataaaag gggcacattt atggtaaatt 2100
gtaaattttt atatatgtat ctttttttca ttttatgcct gagccctgcg agtcactttc 2160
cctttgcttt ttatcaattt tttttttcat cttctttaat ttctttggag ccaagttgga 2220
ctcaaattga ccaagagtct tggatcgcaa agattcaggg atatataagc atagggtcct 2280
tccacctctt ccactttcct tttaattttg ttgcctttta gcccatccag aagcagcact 2340
tttacacaga gtacaatact cttatcttta tctcttgtgt aaaagaagat ccagaacatc 2400
aatgttgatg ctgaagatta aggagaaggt ttagtaaata gatacaaaaa gaaaagtact 2460
gacaaataag cttgtttgca gttgtacctg ggcattgtgc ccacagctgt cgtgatcatc 2520
acaggcgtct tctcatatta ccaagaaagc aagagctccc gtattatgga atctttcaag 2580
aacatggtcc ctcagtatgc tattgttctt cgagatggcg agaagcagaa tgttcaggct 2640
gaggaactgt gcataggaga cattgtagag gtcaagtttg gtgatcgtat cccagctgat 2700
atccgtgtca tcgaaagcag gggcttcaag gttagttcct ggtcttgtat aattgtaata 2760
ttatcattag cagtaaagtt tgtcataaga tttttggcat ttgattcata aaatattagt 2820
tttggtaata gtaatacaaa ctttgccttt gtgctacagg ttgacaactc ttccctgact 2880
ggagaatccg aaccccagag ccgatcaccc gaatacactt ccgagaaccc ccttgagacc 2940
aagaacttgg ctttcttctc caccaatgct gtcgagggtg tgttcttgtg ccttatgtat 3000
tgtcattcac atcttttgtg actttcaaac ttggcaatgt atgttctact gatggtgttg 3060
atgattatga aagggtggta gtcactaatt tctgtgttat tttcaggtac ttgcaagggt 3120
atcgttatca tgattggtga caacactgtg atgggtcgta ttgctggttt ggcatctgga 3180
ttggaaactg gtgaaacccc cattgccaag gaaattaccc atttcattca catcattact 3240
ggtgtggctg tgttcttggg tgtgaccttc ttcgttattg ccttcatcct tggtaaagtt 3300
cttagttgaa aatgtttgtt aaacatgtaa ttttagtctg attaaagagt actgcactgt 3360
acttgacaca tttttatatg tggctcagta aatgcaatgt agatatttta cttgaatcca 3420
tgcacttcaa aagatgttat aaacattagg aattgataca gaaattttat tctaatttaa 3480
aactccacaa tttcagggta ccattggttg gatgctgttg tgttcctcat tggtatcatt 3540
gtagccaatg tgcctgaggg tctgctagcc actgtcactg tgtgcttgac tcttactgcc 3600
aagcgcatgg ctgccaagaa ctgccttgta aagaacttgg aggctgtgga aaccctgggt 3660
tccacttcca ccatttgctc tgataagact ggtaccctca cccagaatcg tatgacagta 3720
gcacatatgt ggttcgacaa taccatcatt gaagctgata catctgaaga tcagtctggc 3780
tgccagtatg acaagacctc acaaggctgg aaggctctgt ctagaattgc tgccctctgc 3840
aaccgtgctg aattcaagac tggcatggaa aacactccca tcctgaaacg tgaagtaaac 3900
ggcgatgctt ccgaagctgc tctgctgaag tgtgtagaat tggctgttgg tgatgttaag 3960
ggctggcgtg cacgcaacaa gaaggtatgt gaaattcctt tcaactccac caacaagtac 4020
caagtatcca tccacgagac tgaggataag aacgacccac gataccttgt tgtgatgaag 4080
ggagcccctg agaggatcct ggaacgttgc tcgaccatct acatcaatgg agaggaaaag 4140
gccctcgacg aagaaatgaa ggaagctttc aacaatgcct accttgaatt gggcggcctt 4200
ggagagcgtg tacttggttt ctgtgactac atgctgccaa ctgacaagta ccctcttgga 4260
taccccttcg atgctgatgc tgtgaacttc cctgtccatg gtctgcgctt cgttggtctg 4320
atgtctatga ttgatcctcc tcgtgctgct gtacctgatg ctgtagcaaa gtgcagatct 4380
gcaggtatca aggttatcat ggttactggt gatcacccca tcactgccaa ggctattgcc 4440
aagtctgtag gtatcatctc tgaaggaaac gagactgttg aggacattgc acagaggttg 4500
aacattccca tcaaggaggt tgaccccact gaagcaaagg ctgctgtagt tcacggttct 4560
gaacttcgtg acatgacatc cgagcagttg gatgatgtcc tcctccacca cactgaaatc 4620
gtgtttgccc gtacctcccc acaacagaag ctgatcattg tagaaggttg ccagcgtatg 4680
ggtgccattg tggctgtaac tggtgatggt gtgaatgatt ctcctgccct gaagaaggct 4740
gatattggta agtatttttt tttttcatct agggatacta ctgtattgac atttcatatt 4800
tgatgtggtt atgctgactt atggcgtgaa acatcacaat attcatggta ttaaacgcaa 4860
tcttattctt acaggtgttg ctatgggtat tgctggttct gatgtgtcca agcaagctgc 4920
tgacatgatc ctgttggacg acaactttgc ttccattgtc actggtgttg aagagggcag 4980
acttattttc gacaacctga agaaatccat tgcttacacc ctgacatcta acatccctga 5040
gacctctccc ttcttgttct tcatgattgc ctcagtccca cttcctcttg gaactgtgac 5100
catcctctgc gttgatctgg gtactgacat ggtaagttat gcaacctttt attctccaag 5160
acaattttca agggaagggt gacactcgac ttttttttct aagacccgca gaacaatatt 5220
ttacttccaa gaataagatc cactcaaaaa taaaaaataa aacacatggc cagacccaag 5280
ataccttttg ccacatttca tatacaaaca tatattaccc tgggtttaca tactgttgaa 5340
taatattttt agcaatcatg gatttttttc ttcccaagct gccagctata tccttggctt 5400
atgaacaagc agaaaatgac ataatgaaga ggcgccccag agatcctata tatgacaaat 5460
tggttaatga gaggtactga gtcaaactgt gatattacat ctgtagtaga tgaaagaatc 5520
gcatagttgt aaagtaattg ccaggtaatt cttgaaaaga aaattattct gtatagtcag 5580
ttttgtaagt catattcaac caaagcttag tagcaagaag taagcatata gtagagtatc 5640
ttaacttttt taaagttgaa aagtcttaga tttgcctctt tacacaccct acaagttcat 5700
tccctgtaca atttccgtcc tctcggtaga tcccagccat atcactggcc tatgaagctg 5760
cggagagtga tatcatgaaa cgatatcccc gaaatccatt caaagacaaa cttgtcaacg 5820
aaaggtaaaa agggctgcat aggaatggat aacatggaac gaatttttta tccagctttt 5880
gcaagatggc aaatttcata ctctgccact tttattagca cgcaaaaatt ggttatggga 5940
tttttctcat ctaataggca aaggtttggt ctcaaatatg aaaagaccat gttttgcatg 6000
tttctttttt atacaatttt ttaaatattg aagagaagaa taagaaaaga agagtggaca 6060
tttttagtaa ggtattttta atattaccag actgttgaaa gaccaacaat ctgaattctc 6120
ttggcttatc caactgggct ggcggggggt aaaaagcaag agtatgcatt ttcccccttt 6180
tttatttcat attttatcta tatttataaa ttataccacg aaagcaggtt tatgagaatg 6240
atcttgctag tattttttta atccccctat taaatgatga tatgaaagtt atgaaaattt 6300
aataactttt actttaagct agctacttca ttattgctta ttctaatact tcaaaatttt 6360
ctcctatgcc tcaccatatg ctaaatttca tcattcactg tgatcaggca atcacatcct 6420
tttattgtta acacattcac acactacggt catatgtgta tgcttgtatt ttttgaaaac 6480
ctgtatatac atatgtatgc acatatctaa gcacacaaaa aaatactcga acatttccac 6540
agttctctac taatcatgca agtgcatttt gcaacatgac aaaatctttc atcaagttct 6601
ttgcttccca tttccttccc ttctacccag cttctaccct ttttattgtt cataaacgat 6660
ggtaaaatca atattttcat tatcagtatt aaggttattg ctgttaatgg atagtggccc 6720
aaacataact gaatagtttt tttttttttt tttttttttt ttttgagaac tggaatgcac 6780
tttttctttt tcataaattt tcctccagag tgcaagtcac aatatgtgat ttttgactga 6840
taatttccaa tttttatatt tatattgaaa catgtttcaa acttttgaag aaatacatca 6900
agggttttgg ttagaccata ttaccatatt tgtttttaga atattatata taaatgcccc 6960
tgcccatcac attgagtaat ctgcacttat caaaactgat actgtttttt caccactgtc 7020
tgggattatt catgaatttc attcataatg tcatcttgca cacggatcac cctgccactg 7080
caggctgagt gtgtagactt gctaactctc agccgtccat atcgcacaac ttgggccgtg 7140
ttcagtaaag tgtatttttc aggccatata gatagttttt cgttttttcc tggattaaat 7200
tttaatatga aagcatgctg gtctctatat tttcatatca gtttttatct ttatacgaaa 7260
aataagttaa aaggcctgaa ggttgcacct gttgcaagcg gtgtttgtgt gtattgacct 7320
gccatgcatg ccatattcat caccatagtg tcgtgcctca aagagttcac cccgtgtgta 7380
tctggtctca tatctgtctt tttttgtctg cctcttgtcc tgtgctggcc atgacaggtc 7440
ccagccattt cccttgccta tgaagcagct gaatctgata ttatgaagcg tcaaccccga 7500
aatccgttca ccgacaagct tgtgaacgag aggtaacgtg tgcaggttct ctctctctct 7560
ctcacacact ttctctctgc ctttctctct ccccctattt atacatatca cttcctgtat 7620
acccatccta caccctaata tcagatcaat caacccagat gctggagaat ctcagtgatc 7680
aaacccttgc atgaatgttc tctagcggat gcagatcctg agtatgtaca agcatcagct 7740
gtgctagctc ctaaaaacga accatacaag gacaaagtcc aagcaatcag gaactagaac 7800
aagaagcaca gcttgcttaa gtactctttt ttgtgggaca cgatgactga ggcttgtgta 7860
ttccatttgt ctggagtata gcaggtccaa attcatcttg tctcatgttt tagcctatat 7920
ccgagatccc ctacacacaa atttactgct gttcaagggg cctgtcacca tctttctggt 7980
aatgctagca catgcccaac aggacagagc agagctcata tcttggtaca tttattgcat 8040
tttattttat ttctaagtgc attttaagat tccaggttat gcaagctgca gcatttgtag 8100
tgactacctt tttacctttt tacgttttct gtcattttcg tcttaagttt tgcatttcat 8160
ttttgtcttg tttaattcta ttttaacctc attatttggt taatatttag gtgcctgcca 8220
tttcccttgc ctatgaagaa gctgagtctg atattatgaa gcgccaaccc cgaaacccat 8280
tcaccgacaa gcttgtgaac gagaggtaat gtttgtataa agtgcagcaa tttgcatact 8340
aacaaattga cgtgtttctc ctcgcgtgaa gtgagttagt gaaatccaac taacaagctt 8400
gttgtcgaaa atatggtggt ggtataacgg tttgaaaaaa ttctttttat tttgaaattg 8460
tgtgaccttt tctctttcta aaggtttcac aaagcttttc ataatgaggg tttcattgtt 8520
ttttcttttt cttttttctt cttacttttt tttgcatgtt tgctttcgtc tcaattgttg 8580
gagcaaatcg agaaaaattg accaacaatt taactttgtg gaagtatggg caaggtcgtt 8640
tagtttaccc acttatttcg tcctctattt gaagttctgt tgtggaactt tcattttatt 8700
ttttttatgg ggcgggtgat tttttcttag aatattacga ctacaaaacc ctttcatggt 8760
ctttgatttt atatagcagt gaggattttg aaatatcagt cctctatact gtattttgtt 8820
ttcttcagtt gagcatatga aaaatcttcg ttggaataac tgatgcatga tttggaaaga 8880
gctttatgtg catggtgtgc ctatttcgcc tttcgtttga ttttcccttt tttactttta 8940
catcctgttc tccttcatcc atgatgcttt ccgtgtccaa agactgttcc aattgcttcg 9000
aaggatatat tccgaaactg ctatggttca taccatgggt cctgtgttcc agccatgtac 9060
tgtagaagtg cacagtagcg tgaatgacgc ggcgataacg gcagtatgca attcggctgc 9120
tcagatgctt cctattaaat gggctgtatt acttaggaaa ttgtctgtgt ctgtctcagt 9180
ctgacttaca atgtaagtag atgatgcttc tagaagttga aatgcaatgc gtatgatcca 9240
tctacatagc cacaccagtc ggtggtcttc actgtgggaa gaggtcgttg ctaggctgac 9300
tggcttttca tgctagatac agggggaaaa agggatggtt ctagtctatt ttacggctcc 9360
tgccttgccc tctacccacc tatgaaccac cacggcgtgt gggctaacca catcaccgat 9420
tgataggggc cagctgagta attgttcatc gccacaccat cctcatggaa gcattcctgg 9480
atgcccgtga gcaacgctaa ttttccgtaa atttttacac aaatgcgcag gtcccggcca 9540
tttccttggc ctatgaggct gcagaatcgg acattatgaa gcgccaacca cgagacccgt 9600
ggcacgacaa acttgtcaat tacaggtgag ctgccaccac tatacaagag gtttttcgca 9660
agcccattct cccttgttac tgtatatcaa gaatgtttgg ttggtgtcaa ggtgaagcat 9720
actgctccca ttggagagct gtaattgatg ttttagaatt ttcaccaaga aaatgtatgt 9780
gttcggacta tgtttacaga caggcgcatc aatacattgc acagaactac ctaatcttac 9840
actcgttggt caatgtttta tggcttaggt ttatgtatat ctatacctta tccatggaat 9900
gccagctctc agcctatact acagtttgtt tgtaattgct gaagccttca ttgtttgata 9960
gtcagtgttc tctaaaaatg tacattgtgg cacatcatac aaggatgtgg atgtaggccg 10020
agtagagacg tcaacaaata atgtgctaca tttccataca taggcaggta cattaatgat 10080
ttattttaca tttgtgattt gtccagtcca tggatcatta gtggcccaac tgtaaaggat 10140
aactacactt tgtcagtatc ttcttctacc tgctttgttt atacaggagt ccttcatgca 10200
ccatgtttgt gagttaactg ggataagaat tttccagtgg atttgtgttc gcactgcgtt 10260
tgtatctttt ctttttgtca tggactgata aggcactctt tatccatacc tcaaacagtt 10320
tgtgaaagtt acttgcccca acatttgtac aaacttctgt ttccctatgc atggaatcta 10380
aggaaaatga atgcatgtat cttgttttgt tttctctgta aaaaatgaaa tgttccacca 10440
ccatagagga tctacagcat caaagaaaag ggtaagggaa tcttatctat gcagataccc 10500
catagagatt aaacctgatt tttcttgtag aattcttact cttagctaag gttttattca 10560
ctacaggata ctatgactaa ccctaagtat ttactctgat tattgtagat agtatgttta 10620
tacatagttc aagtttatat cctactgcta gatgcctttt aattgcagtc ttgcaatttt 10680
cagaagggat ttcgtcagat gacaaaaatt gtttctatta ggtatttggt tgatgtgatt 10740
atctcttagt atgacctttt taagtagata aaatttacat tctttgaatg tttaaaatgt 10800
tcatatatat aattgatatc aggagcactt tttattgatg tgcattattt caccacaact 10860
tttctataat ctccagatag aactcaggaa tatagtatgt cacagttctc tggcatctcc 10920
ttcagttatg caaactacaa tttgtatggg gtttaacttc tttacgaaaa tataaaagat 10980
ttctttcgca tatatattgg ggcaaaacac acatgccata ccagtgtccg tgagttttaa 11040
ttgtatgttg tattccccac acaacaggct catctcaatg gcctatggtc agattggtat 11100
gatccaagcc ctggcaggat tcttcaccta tttcgtgatc atggctgaga acggcttcct 11160
gccaccccat ctctttggtc tccgtgagcg ctgggacagc aaggccatca acgatctgga 11220
ggatcactat ggacaggaat gggtgagttt ctggtttggt ggtgttcatc caagggcgtt 11280
tttgtgcagc ccttttttta ttagttagct tttaaatgta tggacatagc tcaaatttat 11340
ggaagacgag atatgtataa tagaatggtt tttagaaggt aaatatactt gtgtcatgag 11400
tactttactc ttttttccaa cagaccttcc acgaccgtaa gattcttgag tacacctgcc 11460
acactgcttt cttcacctct attgtgattg tgcagtgggc cgatttgatc atttgcaaga 11520
cccgccgtaa ctccattgtc caccagggca tgaagtaagt actgaaaaac tcgacaaaac 11580
ctttcttcgg tagtaggccc gaaaatgtga tcatttatct attattcaag tatcaaatgg 11640
agaagcttgt cagcaaatct tttacaatga atatttgtca gtatatattt atttacttac 11700
cataattact tccaggaact gggtgctgaa ctttggtctc gtctttgaaa ccactttggc 11760
tgccttcctt tcctacaccc caggcatgga caagggtctt cgcatgtacc cactgaagtt 11820
ctactggtgg ctgcctgctc ttccgttctc catccttatc ttcatctacg atgagatacg 11880
tcgcttcatc ctgcgtagga accctggtgg ttggatggaa ctggagacct actattaagc 11940
tgttactgtg agcgcaagaa ttactagagc tcaagactga tcgcatggct cgcatcacct 12000
gccatcatca gtgaacttcc cacagccagc acttgtgtcg ctattacttg tggaaattta 12060
tttaatatcc caatatggaa attaatttct agttgacccc aagaatattt agtaaaagaa 12120
gaggcttggt ctcttcatga ttccatatgt atttcattgt tacttactga ggatattaat 12180
gttattatgg ctgtttctac tttacccatt gttatattta aagtttccag taagtaacag 12240
tgacatacag ctatatatat atatatataa tgtacagaat cctggtgaag gatgtaataa 12300
agacaaaacc gacacttcaa gactgcacca gaagtaattt taaccgtggt ttatataacc 12360
tggtcaacta ctgctccata ctgcaggtgt gagaaagcag tttctcttga aggggggtgg 12420
gaagggataa aatgtttttg aatgctgtaa ac 12452
<210>2
<211>12452
<212>DNA
<213> Litopenaeus vannamei (Litopenaeus vannamei)
<400>2
gtcgtgaagc gcgcgtcgtc gagatcggac gtgccgctgt gccctttagt gtcgaccaac 60
aaacacttca cagacggtga agaatgtgct tgtagcgaaa acctacacag gtcctagagg 120
gcaacattaa gggagtgaac gagtgtacaa agtgaagcct agtggcttgt acataagtgt 180
gtatatacag tgattgttgg tgctggtttg agagccagcg cgtcgccgtt gttcgtggat 240
actgtgtatc ctctgctgcc gtcctcctgc tgagcgccga gtgtcgcctc ttcagcagcc 300
atggccgata cgggcagaac agattcgtat cgacacgcca cggatcgtac catccccgac 360
gataaccgaa cagtgaaagg ggatcctaag tctaagaaaa agccccagaa ggctaaaggg 420
aagaagggag ataaggattt gaatgatctg aagcaggagt tggaacttga tgagcacaag 480
gtcccaattg aggaactctt tcaacgtctc actgttaacc cagacacagt aggtttaccc 540
gagttctttg tagtatcctt ggtatattgc ctaaattaag agtattggta agatttaatc 600
caatagcgac agggaaggaa ttttgggaaa gtgggttaag gagaagtgta cattagcgtg 660
cacagctctg crcattgggt gattctccag ctattttttg taattacttg ytatgattta 720
atctaaaatc acctgtcgct aataggttag artagatctt taagagtaga tamacgcaga 780
taacyctcaa aaatawatca aatgtaattc ttwcttgtgt actttaatta caaggtgttt 840
ttctgycttg aacatgactr accaytccta taaccttgca gggtctatca caaagtgagg 900
cyaagcgccg tattgaacga gatgggccga atgctcttac cccacccaag cagactccag 960
aatgggtcaa gttctgcaaa aacctcttcg gtggtttctc actcctgctg tggattggcg 1020
ctatcctctg cttcattgcc tactcaattg agacagctgc agaagaggag cccaacaagg 1080
acaatgtaag ttttcagtgt tttgtwggaa ataagagtat tctttatcta aagagatgat 1140
tyggtaaatg ttaayatgtt gaaaaatatg tacaactaag ggattcaagt gatttgcaat 1200
tttaaarttr aawtgtagtt atttaatgtt tttcttttga ttaaggtagt tcatgagaaa 1260
tgtcatactt tgttttaggc caattgtttg aaaatgcatt aggcatagca atatatacat 1320
aatcatatct ataagggcat ttgcatttaa caaacttttt cttccttaag ggtttaattt 1380
gacttgcagc tggtatgcag atgttggagt taaaacattt taatggaccg tgtttwgtkt 1440
atttcaatat ccaattcttt ttattagata atgaaactgc tcccatastt aggtttagag 1500
agaaaaatgt tttattaata gatgtgagga agcataaagt twaaagktgt gcaggttaag 1560
aattttagag aatataaatc ataataaatg aaagctagat atttctgagt gttagtgtgt 1620
ctcaaccaat ttatcaaagt tcgtaacatc tyatggtgat attttacagt tatacctagg 1680
tgttgtgctg actggtgtgg tgattataac aggcgtcttc tcctactatc aggaggcaaa 1740
gtcctctgct attatggatt catttaagaa catggtgcct caggtaaacc cttagtaatg 1800
ctacatgcct tagttagcta gatgttggtt ctcctctact gttctgatga graggaggaa 1860
ggtcctctga tgtgtagtgg gtaggtatgg gatcaatcat ttgaggtaag gttagagaat 1920
tagaccctgg gggagaaact aaacacggat gacwtcagag gcttcaaagt cagtgagctm 1980
atagrtagat ggcacctttt ttttttttct tcttcttctt cttcttcctc catgtgaggt 2040
aaggtcatag gaaggaatga tggtctggag tttataaaag gggcacattt atggtaaatt 2100
gtaaattttt atatatgtat ctttttttca ttttatgcct gagccctgcg agtcactttc 2160
cctttgcttt ttatcaattt tttttttcat cttctttaat ttctttggag ccaagttgga 2220
ctcaaattga ccaagagtct tggatcgcaa agattcaggg atatataagc atagggtcct 2280
tccacctctt ccactttcct tttaattttg ttgcctttta gcccatccag aagcagcact 2340
tttacacaga gtacaatact cttatcttta tctcttgtgt aaaagaagat ccagaacatc 2400
aatgttgatg ctgaagatta aggagaaggt ttagtaaata gatacaaaaa gaaaagtact 2460
gacaaataag cttgtttgca gttgtacctg ggcattgtgc ccacagctgt cgtgatcatc 2520
acaggcgtct tctcatatta ccaagaaagc aagagctccc gtattatgga atctttcaag 2580
aacatggtcc ctcagtatgc tattgttctt cgagatggcg agaagcagaa tgttcaggct 2640
gaggaactgt gcataggaga cattgtagag gtcaagtttg gtgatcgtat cccagctgat 2700
atccgtgtca tcgaaagcag gggcttcaag gttagttcct ggwcttgtat aaktgtaata 2760
ttatcattag cagtaaagtt tgtcataaga tttttggcat ttgattcata aaatattagt 2820
tttggtaata gtaatacaaa ctttgccttt gtgcyacagg ttgacaactc ttccctgact 2880
ggagaatccg aaccccagag ccgatcaccc gaatacactt ccgagaaccc ccttgagacc 2940
aagaacttgg ctttcttctc caccaatgct gtcgagggtg tgttcttgtg ccttatgtat 3000
tgtcattcac atcttttgtg actttcaaac tygrcaatgt atgttctact gatggtgttg 3060
atgattatga aagggtgrta gtcactaatt tctgtgttat tttcaggtac ttgcaagggt 3120
atcgttatca tgattggtga caacactgtg atgggtcgta ttgctggttt ggcatctgga 3180
ttggaaactg gtgaaacccc cattgccaag gaaattaccc atttcattca catcattact 3240
ggtgtggctg tgttcttggg tgtgaccttc ttcgttattg ccttcatcct tggtaaagtt 3300
cttagttgaa aatgtttgtt aaacawgtaa ttttagtctg attaaagagt actgcactgt 3360
acttgacmca tttttatatg tggctcagta aatgcaatgt agatatttta cttgaatcca 3420
tgcacttcaa aagatgttat aaacattagg aattgataca gaaatwttat tctaatttaa 3480
aactccacaa tttcagggta ccattggttg gatgctgttg tgttcctcat tggtatcatt 3540
gtagccaatg tgcctgaggg tctgctagcc actgtcactg tgtgcttgac tcttactgcc 3600
aagcgcatgg ctgccaagaa ctgccttgta aagaacttgg aggctgtgga aaccctgggt 3660
tccacttcca ccatttgctc tgataagact ggtaccctca cccagaatcg tatgacagta 3720
gcacatatgt ggttcgacaa taccatcatt gaagctgata catctgaaga tcagtctggc 3780
tgccagtatg acaagacctc acaaggctgg aaggctctgt ctagaattgc tgccctctgc 3840
aaccgtgctg aattcaagac tggcatggaa aacactccca tcctgaaacg tgaagtaaac 3900
ggcgatgctt cygaagctgc tctgctgaag tgtgtagaat tggctgttgg tgatgttaag 3960
ggctggcgtg cacgcaacaa gaaggtatgt gaaattcctt tcaactccac caacaagtac 4020
caagtatcya tccacgagac ygaggataag aacgacccac gataccttgt tgtgatgaag 4080
ggagcccctg agaggatcct ggaacgttgc tcgaccatct acatcaatgg agaggaaaag 4140
gccctcgacg aagaaatgaa ggaagctttc aacaatgcct accttgaatt gggcggcctt 4200
ggagagcgtg tacttggttt ctgtgactac atgctgccaa ctgacaagta ccctcttgga 4260
taccccttcg atgctgatgc tgtgaacttc cctgtccatg gtctgcgctt cgttggtctg 4320
atgtctatga ttgatcctcc tcgtgctgct gtacctgatg ctgtagcaaa gtgcagatct 4380
gcaggtatca aggttatcat ggttactggt gatcacccca tcactgccaa ggctattgcc 4440
aagtctgtag gtatcatctc tgaaggaaac gagactgttg aggacattgc acagaggttg 4500
aacattccca tcaaggaggt tgaccccact gaagcaaagg ctgctgtrgt tcacggttct 4560
gaacttcgtg acatgacatc cgagcagttg gatgatgtcc tcctccacca cactgaaatc 4620
gtgtttgccc gtacctcccc acaacagaag ctgatcattg tagaaggttg ccagcgyatg 4680
ggtgccattg tggctgtaac tggtgatggt gtgaatgatt ctcctgccct gaagaaggct 4740
gatattggta agtwtttttt tttttcatct agggatacta ctgtattgac atttcatatt 4800
tgatgtggtt atgctgactt atggcgtgaa acatcacaat attcatggta ttaaacgcaa 4860
tcttattctt acaggtgttg ctatgggtat tgctggttct gatgtgtcca agcaagctgc 4920
tgacatgatc ctgttggacg acaactttgc ttccattgtc actggtgttg aagagggcag 4980
acttattttc gacaacctga agaaatccat tgcttacacc ctgacatcta acatccctga 5040
gacctctccc ttcttgttct tcatgattgc ctcagtccca cttcctcttg gaactgtgac 5100
catcctctgc gttgatctgg gtactgacat ggtaagttat gcaacctttt attctccaag 5160
acaattttca agggaagggt gacactcgac ttttttttct aagacccgca gaacaatatt 5220
wwacttccaa gaataagatc cactcaaaaa taaaaaataa aacacatggc cagacccaag 5280
ataccttttg ccacatttca tatacaaaca tatattaccc tgggtttaca tactgttgaa 5340
taatattttt agcaatcatg gatttttttc ttcccaagct gccagctata tccttggctt 5400
atgaacaagc agaaaatgac ataatgaaga ggcgccccag agatcctata tatgacaaat 5460
tggttaatga gaggtactga gtcaaactgt gatattacat ctgtagtaga tgaaagaatc 5520
gcatagttgt aaagtaattg ccaggtaatt cttgaaaaga aaattattct gtatagtcag 5580
ttttgtaagt catattcaac caaagcttag tagcaagaag taagcatata gtagagtatc 5640
ytaacttttw taaagttgaa aagtcttaga tttgcctctt tacacaccct acaagttcat 5700
tccctgtaca atttccgtcc tctcggtaga tcccagccat atcactggcc tatgaagctg 5760
cggagagtga tatcatgaaa cgatatcccc gaaatccatt caaagacaaa cttgtcaacg 5820
aaaggtaaaa agggctgcat aggaatggat aacatggaac gaatttttta tccagctttt 5880
gcaagatggc aaatttcata ctctgccact tttattagca cgcaaaaatt ggttatggga 5940
tttttctcat ctaataggca aaggtttggt ctcaaatatg aaaagaccat gttttgcatg 6000
tttctttttt atacaatttt ttaaatattg aagagaagaa taagaaaaga agagtggaca 6060
tttttagtaa ggtattttta atattaccag actgttgaaa gaccaacaat ctgaattctc 6120
ttggcttatc caactgggct ggcggggggt aaaaagcaag agtatgcatt ttcccccttt 6180
tttatttcat attttatcta tatttataaa ttataccacg aaagcaggtt tatgagaatg 6240
atcttgctag tattttttta atccccctat taaatgatga tatgaaagtt atgaaaattt 6300
aataactttt actttaagct agctacttca ttattgctta ttctaatact tcaaaatttt 6360
ctcctatgcc tcaccatatg ctaaatttca tcattcactg tgatcaggca atcacatcct 6420
tttattgtta acacattcac acactacggt catatgtgta tgcttgtatt ttttgaaaac 6480
ctgtatatac atatgtatgc acatatctaa gcacacaaaa aaatactcga acatttccac 6540
agttctctac taatcatgca agtgcatttt gcaacatgac aaaatctttc atcaagttct 6601
ttgcttccca tttccttccc ttctacccag cttctaccct ttttattgtt cataaacgat 6660
ggtaaaatca atattttcat tatcagtatt aaggttattg ctgttaatgg atagtggccc 6720
aaacataact gaatagtttt tttttttttt tttttttttt ttttgagaac tggaatgcac 6780
tttttctttt tcataaattt tcctccagag tgcaagtcac aatatgtgat ttttgactga 6840
taatttccaa tttttatatt tatattgaaa catgtttcaa acttttgaag aaatacatca 6900
agggttttgg ttagaccata ttaccatatt tgtttttaga atattatata taaatgcccc 6960
tgcccatcac attgagtaat ctgcacttat caaaactgat actgtttttt caccactgtc 7020
tgggattatt catgaatttc attcataatg tcatcttgca cacggatcac cctgccactg 7080
caggctgagt gtgtagactt gctaactctc agccgtccat atcgcacaac ttgggccgtg 7140
ttcagtaaag tgtatttttc aggccatata gatagttttt cgttttttcc tggattaaat 7200
tttaatatga aagcatgctg gtctctatat tttcatatca gtttttatct ttatacgaaa 7260
aataagttaa aaggcctgaa ggttgcacct gttgcaagcg gtgtttgtgt gtattgacct 7320
gccatgcatg ccatattcat caccatagtg tcgtgcctca aagagttcac cccgtgtgta 7380
tctggtctca tatctgtctt tttttgtctg cctcttgtcc tgtgctggcc atgacaggtc 7440
ccagccattt cccttgccta tgaagcagct gaatctgata ttatgaagcg tcaaccccga 7500
aatccgttca ccgacaagct tgtgaacgag aggtaacgtg tgcaggttct ctctctctct 7560
ctcacacact ttctctctgc ctttctctct ccccctattt atacatatca cttcctgtat 7620
acccatccta caccctaata tcagatcaat caacccagat gctggagaat ctcagtgatc 7680
aaacccttgc atgaatgttc tctagcggat gcagatcctg agtatgtaca agcatcagct 7740
gtgctagctc ctaaaaacga accatacaag gacaaagtcc aagcaatcag gaactagaac 7800
aagaagcaca gcttgcttaa gtactctttt ttgtgggaca cgatgactga ggcttgtgta 7860
ttccatttgt ctggagtata gcaggtccaa attcatcttg tctcatgttt tagcctatat 7920
ccgagatccc ctacacacaa atttactgct gttcaagggg cctgtcacca tctttctggt 7980
aatgctagca catgcccaac aggacagagc agagctcata tcttggtaca tttattgcat 8040
tttattttat ttctaagtgc attttaagat tccaggttat gcaagctgca gcatttgtag 8100
tgactacctt tttacctttt tacgttttct gtcattttcg tcttaagttt tgcatttcat 8160
ttttgtcttg tttaattcta ttttaacctc attatttggt taatatttag gtgcctgcca 8220
tttcccttgc ctatgaagaa gctgagtctg atattatgaa gcgccaaccc cgaaacccat 8280
tcaccgacaa gcttgtgaac gagaggtaat gtttgtataa agtgcagcaa tttgcatact 8340
aacaaattga cgtgtttctc ctcgcgtgaa gtgagttagt gaartccaac taacaagctt 8400
gttgtcgaaa atatggtggt ggtataacgg tttgaaaaaa ttctttttat tttraaattg 8460
tgtgaccttt tctctttcta aaggtttcac aaagcttttc ataatgrggg tttcattgtw 8520
ttttcttttt cttttttctt cttacttttt tttgcatgkt tgctttcgtc tcaattgttg 8580
gagcaaatcg agraaaattg accaacaatt taactttgtg gaagtatggg caaggtcgtt 8640
tagtttaccc acttatttcg tcctctattt gaagttctgt tgtggaactt tcattttatt 8700
ttttttatgg ggcgggtgat tttttcttag aatattacga ctacaaaacc ctttcatggt 8760
ctttgatttt atatagcagt gaggattttg aaatakcagt cctctatact gtattttgtt 8820
ttcttcwgtt gagcatayga aaaatcttcg ttggaataac tgatgcatga tttggaaaga 8880
gctttatgtg catggtgtgc ctatttcgcc tttcgtttga ttttcccttt tttactttta 8940
catcctgttc tccttcatcc atgatgcttt ccgtgtccaa agactgttcc aattgcttcg 9000
aaggatatat tccgaaactg ctatggttca taccatgggt cctgtgttcc agccatgtac 9060
tgtagaagtg cacagtagcg tgaatgacgc ggcgataacg gcagtatgca attcggctgc 9120
tcagatgctt cctattaaat gggctgtatt acttaggaaa ttgtctgtgt ctgtctcagt 9180
ctgacttaca atgtaagtag atgatgcttc tagaagttga aatgcaatgc gtatgatcca 9240
tctacatagc cacaccagtc ggtggtcttc actgtgggaa gaggtcgttg ctaggctgac 9300
tggcttttca tgctagatac agggggaaaa agggatggtt ctagtctatt ttacggctcc 9360
tgccttgccc tctacccacc tatgaaccac cacggcgtgt gggctaacca catcaccgat 9420
tgataggggc cagctgagta attgttcatc gccacaccat cctcatggaa gcattcctgg 9480
atgcccgtga gcaacgctaa ttttccgtaa atttttacac aaatgcgcag gtcccggcca 9540
tttccttggc ctatgaggct gcagaatcgg acattatgaa gcgccaacca cgagacccgt 9600
ggcacgacaa acttgtcaat tacaggtgag ctgccaccac tatacaagag gtttttcgca 9660
agcccattct cccttgttac tgtatatcaa gaatgtttgg ttggtgtcaa ggtgaagcat 9720
actgctccca ttggagagct gtaattgatg ttttagaatt ttcaccaaga aaatgtatgt 9780
gttcggacta tgtttacaga caggcgcatc aatacattgc acagaactac ctaatcttac 9840
actcgttggt caatgtttta tggcttaggt ttatgtatat ctatacctta tccatggaat 9900
gccagctctc agcctatact acagtttgtt tgtaattgct gaagccttca ttgtttgata 9960
gtcagtgttc tctaaaaatg tacattgtgg cacatcatac aaggatgtgg atgtaggccg 10020
agtagagacg tcaacaaata atgtgctaca tttccataca taggcaggta cattaatgat 10080
ttattttaca tttgtgattt gtccagtcca tggatcatta gtggcccaac tgtaaaggat 10140
aactacactt tgtcagtatc ttcttctacc tgctttgttt atacaggagt ccttcatgca 10200
ccatgtttgt gagttaactg ggataagaat tttccagtgg atttgtgttc gcactgcgtt 10260
tgtatctttt ctttttgtca tggactgata aggcactctt tatccatacc tcaaacagtt 10320
tgtgaaagtt acttgcccca acatttgtac aaacttctgt ttccctatgc atggaatcta 10380
aggaaaatga atgcatgtat cttgttttgt tttctctgta aaaaatgaaa tgttccacca 10440
ccatagagga tctacagcat caaagaaaag ggtaagggaa tcttatctat gcagataccc 10500
catagagatt aaacctgatt tttcttgtag aattcttact cttagctaag gttttattca 10560
ctacaggata ctatgactaa ccctaagtat ttactctgat tattgtagat agtatgttta 10620
tacatagttc aagtttatat cctactgcta gatgcctttt aattgcagtc ttgcaatttt 10680
cagaagggat ttcgtcagat gacaaaaatt gtttctatta ggtatttggt tgatgtgatt 10740
atctcttagt atgacctttt taagtagata aaatttacat tctttgaatg tttaaaatkt 10800
tcatatatat aattgatatc aggagcactt tttattgatg tgcattattt caccacaact 10860
tttctataat ctccagatag aactcaggaa tatagtatgt cayagttctc tggcatctcc 10920
ttcagttatg caaactacaa twtgtatggg gtttaacttc tttamgaaaa tataaaagat 10980
ttctttcgca tatatattgg ggcaaaayac acatgccata ccagtgtcck tgagttttaa 11040
ttgtatgttg tattccmcac acaacaggct catctcaatg gcctatggtc agattggtat 11100
gatccaagcc ctggcaggat tcttcaccta tttcgtgatc atggctgaga acggcttcct 11160
gccaccccat ctctttggtc tccgtgagcg ctgggacagc aaggccatca acgatcwgga 11220
ggatcactat ggacaggaat gggtgagttt ctggtttggt ggtgttyatc cragggcgtt 11280
tttgtgcaky cctttttyww ttagttagyt tktaaatgta tggacatagc tcaaatttat 11340
ggaagacgag atatgtataa tagaatggtt tttaraaggt aaatataytt gtgtcatgag 11400
tactttactc ttttttccaa cagaccttcc acgaccgtaa gattcttgag tacacctgcc 11460
acactgcttt cttcacctct attgtgattg tgcagtgggc cgatttgatc atttgcaaga 11520
cccgccgtaa ctccattgtc caccagggca tgaagtaagt actgaaaaac tcgacaaaac 11580
ctttcttcgg tagtaggccc gaaaatgtga tcatttatct attattcaag tatcaaatgg 11640
agaagcttrt cagcaaatct ttwacaatga atatttgyca gtatatatwt atttacttac 11700
cataattact tccaggaact gggtgctgaa ctttggtctc gtctttgaaa ccactttggc 11760
tgccttcctt tcctacaccc caggcatgga caagggtctt cgcatgtacc cactgaagtt 11820
ctactggtgg ctgcctgctc ttccgttctc catccttatc ttcatctacg atgagatacg 11880
tcgcttcatc ctgcgtagga accctggtgg ttggatggaa ctggagacct actattaagc 11940
tgttactgtg agcgcaagaa ttactagagc tcaagactga tcgcatggct cgcatcacct 12000
gccatcatca gtgaacttcc cacagccagc acttgtgtcg ctattacttg tggaaattta 12060
tttaatatcc caatatggaa attaatttct agttgacccc aagaatattt agtaaaagaa 12120
gaggcttggt ctcttcatga ttccatatgt atttcattgt tacttactga ggatattaat 12180
gttattatgg ctgtttctac tttacccatt gttatattta aagtttccag taagtaacag 12240
tgacatacag ctatatatat atatatataa tgtacagaat cctggtgaag gatgtaataa 12300
agacaaaacc gacacttcaa gactgcacca gaagtaattt taaccgtggt ttatataacc 12360
tggtcaacta ctgctccata ctgcaggtgt gagaaagcag tttctcttga aggggggtgg 12420
gaagggataa aatgtttttg aatgctgtaa ac 12452
Claims (5)
1. An amplification primer for screening the SNP marker of a Na, K-ATPase alpha subunit gene of a litopenaeus vannamei, which is characterized by comprising 13 pairs of amplification primers:
a primer pair 13: LvNK001-LvNK 011-F: 5'-TGGAACTTGATGAGCACAA-3', respectively;
LvNK001-LvNK011-R：5’-GGCAATGAAGCAGAGGATA-3’；
primer pair 14: LvNK012-LvNK 023-F: 5'-CTCCTGCTGTGGATTGGC-3', respectively;
LvNK012-LvNK023-R：5’-GACTTTGCCTCCTGATAG-3’；
primer pair 15: LvNK024-LvNK 027-F: 5'-GGCGTCTTCTCCTACTATCA-3', respectively;
LvNK024-LvNK027-R：5’-AAAGCAAAGGGAAAGTGA-3’；
primer pair 16: LvNK028-LvNK 030-F: 5'-GCAGTTGTACCTGGGCATTG-3', respectively;
LvNK028-LvNK030-R：5’-GACAGCATTGGTGGAGAA-3’；
a primer pair 17: LvNK031-LvNK 036-F: 5'-TTCTCCACCAATGCTGTC-3', respectively;
LvNK031-LvNK036-R：5’-GGCTACAATGATACCAATGAG-3’；
a primer pair 18: LvNK037-LvNK 039-F: 5'-TGTTGTGTTCCTCATTGGT-3', respectively;
LvNK037-LvNK039-R：5’-AGGTAGGCATTGTTGAAAG-3’；
primer pair 19: LvNK040-LvNK 042-F: 5'-ATTCCCATCAAGGAGGTT-3', respectively;
LvNK040-LvNK042-R：5’-GGATGTTAGATGTCAGGGT-3’；
and (3) primer pair 20: LvNK043-LvNK 046-F: 5'-CATTGCTTACACCCTGAC-3', respectively;
LvNK043-LvNK046-R：5’-CAGGGAATGAACTTGTAGG-3’；
a primer pair 21: LvNK047-LvNK 052-F: 5'-TATTTAGGTGCCTGCCATTT-3', respectively;
LvNK047-LvNK052-R：5’-AGGACGAAATAAGTGGGT-3’；
primer pair 22: LvNK053-LvNK 055-F: 5'-TCGTTTAGTTTACCCACT-3', respectively;
LvNK053-LvNK055-R：5’-TGTGGCTATGTAGATGGA-3’；
primer pair 23: LvNK056-LvNK 063-F: 5'-TTCAGAAGGGATTTCGTC-3', respectively;
LvNK056-LvNK063-R：5’-GTCCATAGTGATCCTCCA-3’；
a primer pair 24: LvNK064-LvNK 074-F: 5'-ACAACAGGCTCATCTCAA-3', respectively;
LvNK064-LvNK074-R：5’-CTGGTGGACAATGGAGTT-3’；
primer pair 25: LvNK075-LvNK 078-F: 5'-GCCGTAACTCCATTGTCC-3', respectively;
LvNK075-LvNK078-R：5’-TGGAGAACGGAAGAGCAG-3’。
2. a method for screening a Na, K-ATPase alpha subunit gene SNP marker of a litopenaeus vannamei is characterized by comprising the following steps: respectively using a plurality of Litopenaeus vannamei genomic DNAs from different sources as templates, and respectively using the primer pair LvNK001-LvNK011-F/LvNK001-LvNK011-R, LvNK-LvNK 023-F/LvNK012-LvNK 023-LvNK-R, LvNK024-LvNK027-F/LvNK024-LvNK027-R, LvNK028-LvNK030-F/LvNK028-LvNK030-R, LvNK-031-LvNK 036-F/LvNK031-LvNK036-R, LvNK037-LvNK039-F/LvNK037-LvNK039-R, LvNK-LvNK 042-F/LvNK 048-LvNK 043-LvNK 048-LvNK 043-0527-LvNK 043-LvNK 053-F/LvNK 053-052-053, LvNK056-LvNK063-F/LvNK056-LvNK063-R, LvNK064-LvNK074-F/LvNK064-LvNK074-R, LvNK075-LvNK078-F/LvNK075-LvNK078-R is used as an amplification primer to carry out PCR amplification to respectively obtain amplification products, then the amplification products are sequenced, sequencing graphs of the amplification products obtained by each pair of primer amplification templates are used as a group to be compared, and SNP sites are screened.
3. The screening method of claim 2, wherein the PCR amplification reaction system is 25 μ L, and comprises: not containing Mg2+10 × PCR buffer 2.5. mu.L, 25mM MgCl2mu.L of 2.0. mu.L, 10mM dNTP 0.5. mu.L, 5U/. mu.L of high fidelity PCR enzyme 0.2. mu.L, 10. mu.M forward primer 0.5. mu.L, 10. mu.M reverse primer 0.5. mu. L, DNA template 12.5ng, the remainder being made up to 25. mu.L with sterile water.
4. The screening method of claim 2 or 3, wherein the PCR amplification is performed by the following reaction procedures: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 1 minute for 35 cycles; the extension was continued for 10 minutes at 72 ℃.
5. The screening method of claim 2, wherein the Na, K-ATPase alpha subunit gene of Litopenaeus vannamei has 78 SNP sites, wherein the nucleotide sequence shown in SEQ ID NO.2 has A or G at the 672 th base, T or C at the 711 th base, A or G at the 752 nd base, A or C at the 773 th base, T or C at the 785 th base, A or T at the 796 th base, A or T at the 813 th base, T or C at the 846 nd base, A or G at the 860 th base, T or C at the 865 th base, T or C at the 902 th base, A or T at the 1106 th base, T or C at the 2 nd base, T or C at the 1155 th base, T or C at the 1207 th base, A or G at the 1210 th base, A or G at the 1213 th base, A or T at the 1436 th base, T or G at the 1439 th base, T or G at the 1143 th base, 1488 base is G or C, 1542 base is A or T, 1547 base is T or G, 1652 base is T or C, 1852 base is A or G, 1954 base is A or T, 1980 base is A or C, 1985 base is A or G, 2743 base is A or T, 2753 base is T or G, 2855 base is T or C, 3032 base is T or C, 3034 base is A or G, 3078 base is A or G, 3326 base is A or T, 3368 base is A or C, 3466 base is A or T, 3912 base is T or C, 4029 base is T or C, 4041 base is T or C, 4548 base is A or G, 4677 base is T or T, 4754 base is T or T, 5241 base is T or C, 5221 base is T or T, 5241 base is A or C, 5241 base is A or G, 527 base is T or C, 5254 base is T or T, A or T5641 base is T, 5641 base is T or T, 5650 th base is A or T, 8384 th base is A or G, 8454 th base is A or G, 8507 th base is A or G, 8520 th base is A or T, 8559 th base is T or G, 8593 th base is A or G, 8796 th base is T or G, 8827 th base is A or T, 8838 th base is T or C, 10799 th base is T or G, 10903 th base is T or C, 10942 th base is A or T, 10965 th base is A or C, 11008 th base is T or C, 11030 th base is T or G, 11057 th base is A or C, 11017 th base is A or T, 11267 th base is T or C, 11272 th base is A or G, 11289 th base is T or G, 1102 th base is T or C, 11298 th base is T or T, 11264 th base is T or 38964 th base, 11300 th base is T or C, 11300 th base is A or G, 11200 th base is T or C, 11300 th base is T or C, 38964 th base, The 11312 th base is T or G, the 11375 th base is A or G, the 11388 th base is T or C, the 11649 th base is A or G, the 11663 th base is A or T, the 11678 th base is T or C, and the 11689 th base is A or T.
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| CN110791571B (en) * | 2019-11-15 | 2021-06-29 | 中国科学院南海海洋研究所 | SNP marker for distinguishing Vibrio harveyi infection resistance of litopenaeus vannamei, and detection method and application thereof |
| CN111041012B (en) * | 2019-12-13 | 2022-04-26 | 中国科学院南海海洋研究所 | Litopenaeus vannamei endoplasmic reticulum Ca-ATP enzyme LvSERCA gene and encoding protein and application thereof |
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