SNP molecular markers related to high ammonia nitrogen tolerance in Litopenaeus vannamei LvInx2 gene and their application

Abstract

This invention discloses a SNP molecular marker related to high ammonia nitrogen tolerance in the LvInx2 gene of Litopenaeus vannamei and its application. The SNP molecular marker is located in the nucleotide sequence of the LvInx2 gene of Litopenaeus vannamei as shown in SEQ ID NO: 1, and the nucleotide sequence shown in SEQ ID NO: 1 has an A / G base polymorphism at position 680 from the 5' end. This invention also provides primers for amplifying the SNP molecular marker and kits and methods for detecting the SNP molecular marker; as well as the application of reagents, primers, kits or detection methods for detecting the SNP molecular marker in identifying or breeding Litopenaeus vannamei varieties with high ammonia nitrogen tolerance. In the genetic improvement of Litopenaeus vannamei, individuals with the AA genotype at the 680A>G site can be preferentially selected as superior parents. This scheme provides effective molecular tools and technical support for the precise breeding of new Litopenaeus vannamei varieties tolerant to high ammonia nitrogen.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a molecular marker for the high ammonia nitrogen tolerance trait of the LvInx2 gene in Litopenaeus vannamei and its application. Background Technology

[0002] Ammonia nitrogen, as a typical environmental stressor in aquaculture ecosystems, has become a focus of aquatic toxicology research due to its toxicological effects. Because it lacks a charge, non-ionic ammonia has strong permeability and is more easily dissolved in lipids, causing damage to tissues such as the gills, hepatopancreas, and intestines of shrimp, making them more susceptible to ammonia toxicity. Ammonia nitrogen stress inhibits the immune defense function of crustaceans through multiple pathways. For example, ammonia nitrogen exposure can disrupt the expression of genes related to intestinal barrier function, promote the production of inflammatory cytokines, disrupt intestinal immune homeostasis, reduce beneficial bacteria and increase pathogenic bacteria in the gut, and impair the normal digestive and immune functions of crustaceans. Elucidating the genetic and molecular mechanisms of ammonia nitrogen tolerance in shrimp has become an important goal for the healthy development of aquaculture.

[0003] Currently, marker-assisted selection breeding technology has become a key means of improving the stress resistance traits of species. Among them, single nucleotide polymorphisms (SNPs), as third-generation molecular markers, are widely used in animal and plant breeding due to their advantages such as abundant quantity, high stability, and ease of high-throughput detection. However, in the field of genetic improvement of Litopenaeus vannamei, especially for the important trait of high ammonia nitrogen tolerance, the development of relevant SNP markers is still lagging behind, and their application in actual breeding is even weaker, which restricts the efficient breeding of high ammonia nitrogen tolerance traits in shrimp.

[0004] Therefore, this invention aims to develop an SNP marker that is significantly associated with the gap junction protein LvInx2 (Litopenaeus vannamei innexin inx2, LvInx2) gene in Litopenaeus vannamei. This marker can be used for marker-assisted breeding to efficiently select for high ammonia nitrogen tolerance, thereby improving the environmental adaptability, economic benefits, and industrial sustainability of shrimp farming. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular marker for the high ammonia nitrogen tolerance trait of the LvInx2 gene in Litopenaeus vannamei, primers for amplifying the molecular marker, and a kit for detecting the molecular marker.

[0006] The present invention also aims to provide a method for detecting the high ammonia nitrogen tolerance trait of Litopenaeus vannamei.

[0007] The final object of the present invention is to provide the application of reagents, primers, kits or detection methods for detecting the SNP molecular markers in the identification or selection of Litopenaeus vannamei varieties with high ammonia nitrogen tolerance.

[0008] The first objective of the present invention can be achieved by the following technical solution: a SNP molecular marker related to the high ammonia nitrogen tolerance trait of the Litopenaeus vannamei LvInx2 gene, wherein the SNP molecular marker is located in the nucleotide sequence of the Litopenaeus vannamei LvInx2 gene as shown in SEQ ID NO: 1, and the nucleotide sequence shown in SEQ ID NO: 1 is located at position 680 from the 5' end, and its base is A or G.

[0009] The team of the inventors of this application conducted large-scale whole-genome resequencing of Litopenaeus vannamei, aligned the obtained sequencing data with the Litopenaeus vannamei reference genome (NCBI: GCF 003789085.1), and identified and preliminarily screened SNPs using GATK software. Plink2 software was used for quality control filtering of the original variant sites to obtain a high-quality SNP dataset. Based on this, GCTA software was used to perform principal component analysis and assess inter-sample phylogenetic relationships on the screened variant sites. Four statistical models were designed for continuous phenotypes (survival time) and binary phenotypes (ammonia nitrogen sensitivity / tolerance): a simple linear model (LM); a general linear model with population structure as a fixed effect (GLM(Q)); a mixed linear model incorporating a kinship matrix (MLM(K)); and a mixed linear model integrating both population structure and kinship (MLM(QK)). Genome-wide association analysis was performed using these models, and the intersection of the obtained significant variant sites was taken. Finally, a SNP site significantly associated with the high ammonia nitrogen tolerance trait was detected in the LvInx2 gene region (i.e., the 680th nucleotide sequence from the 5' end of the SEQ ID NO: 1, with bases A or G). This site is located in the intron region of the gene.

[0010] The SNP molecular markers described in this invention are significantly correlated with the survival rate of Litopenaeus vannamei under high ammonia nitrogen stress.

[0011] Therefore, this invention focuses on the SNP sites of the LvInx2 gene in Litopenaeus vannamei and found that one SNP site (680A>G) located in the intron region of the LvInx2 gene is significantly associated with the high ammonia nitrogen tolerance trait of Litopenaeus vannamei. Thus, the SNP molecular markers were obtained through screening.

[0012] Further small-group experiments and large-group validations revealed that the SNP sites in the LvInx2 gene of Litopenaeus vannamei were identified as follows:

[0013] Individuals with the AA genotype (680A>G) showed significantly higher tolerance to high ammonia nitrogen than individuals with the GG genotype.

[0014] The genotypes of high ammonia nitrogen tolerance showed significant differences among the SNP sites located in the intron region of the LvInx2 gene in Litopenaeus vannamei (P<0.01).

[0015] That is, the AA genotype individuals with the SNP molecular marker at position 680 of the LvInx2 gene as shown in SEQ ID NO: 1 have significantly higher tolerance to high ammonia nitrogen than the GG genotype individuals.

[0016] The aforementioned high ammonia nitrogen tolerance trait specifically refers to the enhanced survival adaptability of Litopenaeus vannamei in sublethal high ammonia nitrogen stress environments. Under these conditions, the ammonia nitrogen concentration in the water far exceeds the safe threshold for conventional aquaculture, yet individuals possessing this trait can still survive, with their tolerance duration or survival rate significantly superior to that of ordinary populations.

[0017] The present invention also provides primers for amplifying the SNP molecular marker, the primers comprising a forward primer LvInx2-F and a reverse primer LvInx2-R, the nucleotide sequence of the forward primer LvInx2-F being shown in SEQ ID NO: 2, and the nucleotide sequence of the reverse primer LvInx2-R being shown in SEQ ID NO: 3.

[0018] Specifically, the nucleotide sequences of the forward primer LvInx2-F and the reverse primer LvInx2-R are as follows:

[0019] Forward primer LvInx2-F:5'-gctacactgcctggttgtct-3' (as shown in SEQ ID NO: 2);

[0020] Reverse primer LvInx2-R:5'-gggaaaacaaggccgcaaat-3' (as shown in SEQ ID NO: 3).

[0021] The present invention also provides a kit for detecting the SNP molecular marker, the kit comprising the primers.

[0022] The second objective of this invention can be achieved through the following technical solution: a method for detecting the high ammonia nitrogen tolerance trait of Litopenaeus vannamei, comprising the following steps:

[0023] (S1) Extract genomic DNA from the gill tissue of Litopenaeus vannamei;

[0024] (S2) Using the primers described above, the genomic DNA of the gill tissue of Litopenaeus vannamei described in step (S1) is amplified by PCR to obtain the amplification product;

[0025] (S3) Sequencing the amplification products described in step (S2), and determining the genotype of the SNP molecular marker based on the sequencing results;

[0026] (S4) The high ammonia nitrogen tolerance trait of Litopenaeus vannamei is determined by the genotype of the SNP molecular marker described in step (S3); the high ammonia nitrogen tolerance trait of individuals with the AA genotype of the SNP molecular marker is significantly higher than that of individuals with the GG genotype.

[0027] In the above-mentioned detection methods for the high ammonia nitrogen tolerance trait of Litopenaeus vannamei:

[0028] Preferably, the PCR reaction system used in step (S2) for PCR amplification is 20 μL, including: 10 μL of 2×MagicTaqPCR Super(+dye), 1 μL each of 10 mM forward and reverse primers, 1 μL of 40 ng / μL DNA template, and 7 μL of ddH2O.

[0029] Preferably, in step (S2), the PCR amplification process uses a total of 32 cycles of PCR reaction program, with a pre-denaturation of 95 °C for 5 min before each cycle, each cycle including denaturation at 95 °C for 30 sec, annealing at 57 °C for 30 sec, extension at 72 °C for 30 sec, and extension at 72 °C for 10 min after each cycle.

[0030] The present invention provides a method for detecting the high ammonia nitrogen tolerance trait of Litopenaeus vannamei. By detecting the SNP molecular marker genotype of Litopenaeus vannamei, the high ammonia nitrogen tolerance ability of Litopenaeus vannamei is determined based on the detection results of the high ammonia nitrogen tolerance trait.

[0031] The last objective of the present invention can be achieved by the following technical solution: the application of reagents, primers, kits or methods for detecting the SNP molecular marker in the identification or selection of Litopenaeus vannamei varieties with high ammonia nitrogen tolerance; the SNP molecular marker is located in the nucleotide sequence of the Litopenaeus vannamei LvInx2 gene as shown in SEQ ID NO: 1, wherein the nucleotide sequence shown in SEQ ID NO: 1 is at position 680 from the 5' end, and its base is A or G; individuals with the AA genotype of the SNP molecular marker have significantly higher high ammonia nitrogen tolerance than individuals with the GG genotype.

[0032] In summary, this invention discloses a molecular marker for a high ammonia nitrogen tolerance trait in the LvInx2 gene of Litopenaeus vannamei and its application. By screening for high ammonia nitrogen tolerance in Litopenaeus vannamei, this invention identified a SNP site on the LvInx2 gene that is significantly associated with this trait and designed a pair of primers to amplify the SNP site. The disclosed SNP site is significantly correlated with the survival rate of Litopenaeus vannamei under high ammonia nitrogen stress. Individuals with the AA genotype at the SNP site show significantly stronger high ammonia nitrogen tolerance than individuals with other genotypes. This marker can be used for marker-assisted breeding and genetic improvement of Litopenaeus vannamei, enhancing its high ammonia nitrogen tolerance and increasing aquaculture efficiency.

[0033] The beneficial effects of this invention are as follows:

[0034] (1) This invention takes the SNP site of the LvInx2 gene of Litopenaeus vannamei as the research target and finds that a SNP site (680A>G) located in the intron region of the LvInx2 gene is significantly associated with the high ammonia nitrogen tolerance trait of Litopenaeus vannamei.

[0035] (2) In this invention, the AA genotype individuals with 680A>G showed significantly higher tolerance to high ammonia nitrogen than the GG genotype individuals (P<0.01).

[0036] (3) The SNP sites disclosed in this invention are significantly correlated with the survival rate of Litopenaeus vannamei under high ammonia nitrogen stress. Individuals with the genotype AA in the SNP sites have significantly stronger tolerance to high ammonia nitrogen than individuals with other genotypes. They can be used for molecular marker-assisted breeding and genetic improvement of Litopenaeus vannamei to improve the tolerance of Litopenaeus vannamei to high ammonia nitrogen and increase aquaculture benefits.

[0037] (4) In the genetic breeding research of Litopenaeus vannamei with high ammonia nitrogen tolerance as the breeding index, individuals with AA at the 680A>G locus can be selected as breeding parents. This has important guiding significance for the breeding of new varieties of Litopenaeus vannamei with excellent high ammonia nitrogen tolerance. Attached Figure Description

[0038] Figure 1 This is a sequencing peak diagram of the PCR amplification product sequenced using LvInx2-F primers in Example 2. The figure shows the position of the 680A>G site. Detailed Implementation

[0039] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, it should be noted that the following embodiments are only used to describe the content of the invention and do not constitute a limitation on the scope of protection of the present invention. Unless otherwise specified, the reagents or materials used in the embodiments are all from commercial sources. Unless otherwise specified, the experimental instruments used are all conventional laboratory instruments.

[0040] Example 1

[0041] This embodiment utilizes variant sites obtained from whole-genome resequencing. Genome-wide association analysis (GWA) is performed using these variant sites in both continuous phenotype (survival time) and binary phenotype (ammonia nitrogen sensitivity / tolerance). Ultimately, a SNP site significantly associated with high ammonia nitrogen tolerance was detected within the LvInx2 gene region. This site is located at position 680 from the 5' end of the Litopenaeus vannamei LvInx2 gene (as shown in SEQ ID NO: 1), with bases A or G (680A>G). The method for screening the SNP site includes the following steps:

[0042] (1) Acquisition of Litopenaeus vannamei population

[0043] The experiment was conducted at the Donghai Island Marine Biology Research Base of Guangdong Ocean University, China. The shrimp used in the experiment were purchased from a local shrimp farm on Donghai Island, Zhanjiang City, and temporarily housed at the Guangdong Ocean University Marine Biology Research Base. Prior to the experiment, the shrimp underwent a one-week acclimatization period. During this period, optimal water conditions were maintained through continuous aeration and daily water changes. The water temperature was controlled at 25 ± 1℃, and the salinity at 30‰. In addition, dissolved oxygen and ammonia nitrogen levels were monitored daily to ensure these key water quality parameters remained within normal ranges. Commercial feed was administered three times daily.

[0044] (2) High ammonia nitrogen stress experiment and tissue sampling

[0045] One hundred and eighty healthy and vigorous Litopenaeus vannamei shrimp (9.14 ± 1.37 g) were randomly and evenly distributed into nine tanks (20 shrimp per tank), each containing 80 L of fresh seawater. The ammonia nitrogen concentration in the water was adjusted to 50 mg / L by adding ammonium chloride. The premixed fresh seawater with ammonium chloride was replaced daily, and the ammonia nitrogen level in the water was monitored to ensure a constant ammonia nitrogen stress.

[0046] After the experiment began, the time of death for each shrimp was recorded until all shrimp had died. The criterion for determining death was: if a shrimp lay on its side and did not respond to touch. After recording the time of death, the gills of the shrimp were immediately removed under sterile conditions in duplicate, placed in cryovials and stored in liquid nitrogen, and then transferred to an ultra-low temperature freezer at -80 °C until DNA was extracted.

[0047] (3) DNA extraction and whole genome resequencing

[0048] DNA was extracted from gill tissue using the MolPure Cell / Tissue DNA Kit (Yeasen). A whole-genome sequencing library was then constructed, and the resulting library was sequenced on the Illumina X-ten high-throughput sequencing platform.

[0049] (4) Raw data filtering and quality control of variant sites

[0050] The obtained sequencing data were aligned with the Litopenaeus vannamei reference genome (NCBI: GCF 003789085.1), and SNPs were identified and preliminarily screened using GATK software. Plink2 software was used for quality control filtering of the original variant sites to obtain a high-quality SNP dataset. Based on this, GCTA software was used to perform principal component analysis and assess inter-sample phylogenetic relationships on the screened variant sites.

[0051] (5) Genome-wide association analysis of shrimp tolerance to high ammonia nitrogen

[0052] Genome-wide association analyses were performed for both continuous phenotypes (survival time) and binary phenotypes (ammonia sensitivity / tolerance). The analyses were conducted using GCTA software, and the statistical models employed included: simple linear model (LM), general linear model with population structure as a fixed effect (GLM(Q)), mixed linear model incorporating kinship matrix as a random effect (MLM(K)), and mixed linear model integrating both population structure and kinship (MLM(QK)). The expression for the MLM(QK) model is as follows:

[0053] y = Xα + Qβ + Kμ + e

[0054] In this unified expression, y is the phenotypic vector, X is the genotype matrix, α is the genotype effect vector, Q is the fixed-effects matrix, β is the fixed-effects vector (population structure), K is the random-effects matrix (kinship), μ is the random-effects vector, and e is the residual vector. The remaining models are specific combinations of this unified expression. Through the above multi-model association analysis, SNP molecular markers (significance less than 1 × 10⁻⁶) significantly associated with the high ammonia nitrogen tolerance trait in Litopenaeus vannamei were finally identified. -5 ).

[0055] The results showed that a SNP site significantly associated with high ammonia nitrogen tolerance was finally detected in the LvInx2 gene region of Litopenaeus vannamei. This site is located at position 680 from the 5' end of the LvInx2 gene of Litopenaeus vannamei (as shown in SEQ ID NO: 1), with bases A or G (680A>G). It can be used as a molecular marker for screening Litopenaeus vannamei parents for high ammonia nitrogen tolerance-assisted breeding.

[0056] The nucleotide sequence of the LvInx2 gene in Litopenaeus vannamei (as shown in SEQ ID NO: 1) is as follows:

[0057] cccgtgcccc cctccctcct tattctccac acgccactac ctctcactct cactcattcgctctccctcc 70

[0058] ctcatacctt tccctcccgc cctccgtctc tcactcgact tgaccgtgcg tgcgcgcgtgatcgctgact 140

[0059] gacgccggag aaaaagccat gtcaagtaac tgatcctgtc gctgacgtct gggctgcgctgccccgccgc 210

[0060] gcagtgttcg gaaggggcgc tccttcagtg tcgtcgggga tttcaaagca aggacccacacctcgaaaac 280

[0061] gccacctcgg acactccccc ctcatcccct cgccccctcc cgtgtgttta tcgtgtaccaaccgtgtgtg 350

[0062] agcgcagcat gagagcacgg ccgaggacct agcttgcaac gagagagcag ctctgcaggacctgaggatc 420

[0063] ttagcatacg ttacacctga gtcccccgag tcctgttgat agaacttgcg gccaggactccaccgcctcc 490

[0064] gctgccggac gtacgaacgt ttcgcggtgt ggatagtgtc gtacaacctc ttatcagtaaagga gctaca 560

[0065] ctgcctggtt gtct cccagg ccaagagcag ataatagtcg acctaaggac tcttatcagaggtgagtgat 630

[0066] tcccagacgc ggttccttc ttcccattag cgtttcggcg ccagtgtga a cagataggagtgatgttagc 700

[0067] attgttttt tatgctttat ggacgcgagt gtgttgtgct ggaatgagaa gtagggaatggatagaatta 770

[0068] atgatgctct ttggccaaga acaatggctt cccagttttt cctcccccag tctcggttaattcggtagga 840

[0069] aataggtagg cctggtgacg tcattcctttt gaatttggga ggacatactt gcagaagcgatcagctagtt 910

[0070] taccctttgt tctgcgggat cgacccttgc attgttcagg gaatactcgc gtaaatcggggtgt atttgc 980

[0071] ggccttgttt tccc tcctgt ttcctgatat atttagctcc gtatcttttc aagggctatgaatacaaaaa 1050

[0072] aaaaaaata ataagatgca ttgcagaatt aaatggttga gttcccccct cccaccccaccctccccact 1120

[0073] ctcgttttta atacacccct caggctacac attactaata cagagaacga aatacgttacgtcttaattc 1190

[0074] ccggaaaaaa tggaggagtc agtgattttt cttatatata tatacaataa tctttttctttgcacagtctt 1260

[0075] cgtatttacc agcagtgaca gttgccaaat cttgctctc atccttcaat gtaggttactagtagcaaac 1330

[0076] gaaggttata ttacacatct atatatttttttctagttc ttcccatggc atttcttttgtatatatgaa 1400

[0077] ttctcttgtg ctttttttttttttggggggggggggggtt ctgtggcttg tgacgtagaatgtctgtttt 1470

[0078] attatttt attcgtttta gtagtgtggg tgcgtggtct tattatcctt ctggaatttttctttgcgtc 1540

[0079] tctctctctg tttattcatg ttctgttcct attcgtgtaa gggatgtatg tgtgattttttttaaat 1610

[0080] gtaaataatg cgtattgca gagtcgtgcc ttggatcgaa aaatactga ttctttattgtataatt 1680

[0081] ttgcatcgt tgttttcat tgtcatagaa acacaagcgt ctcgtgttgt tgtccattgtgttctctctt 1750

[0082] tataaaaaaactaaat aagtaaaaaaaaaaaaaaaaacata tccgacagttctcgcttcaa 1820

[0083] tggggatttc tggtcctggc ggagtgtgac gtcacaggac gatgtcgaa atattttgatgtttagagaa 1890

[0084] gagagagag gagagagag gagagagag gagagagag gagagagag gagagagag 1960

[0085] gagagagaga gagagactga gagactgagg cttttgttct tcaccaatca acttgtttctgggtccaatt 2030

[0086] ctctcttctg tccgaggga aggagagaa gaatcagaag tatcagaaga aaacgaagaagtagaagaag 2100

[0087] aagaagtagt agaagaagaa gaagtagggag gacgaaag aagtagagaa aaaggaaagcaagacgtttca 2170

[0088] ggctaatagt ttccagtttg gaatcatttc tgggaagga aggaggtggt gggtagtgtggctcagggcg 2240

[0089] tggggtggct gtctggaagg gaggggggg aagggggcgt gtgggcaagg tggggtcgaggagaaggagg 2310

[0090] aagggtgggg ggcaggggt tgaggggggtt gggggtcgag gagaaggagg aagggggtgggggggggttg 2380

[0091] ggggtcgaaga gaaggagaa gggggtgggg agggaggtag ggggtgggg gtggggggaggatagcggtc 2450

[0092] gtgagacggt caaactcaat tccaaaccgc cggaagtgag caacgagaag gtattctgcattcacctgtg 2520

[0093] 2590

[0094] gacgggcgag tagagggt agacgggcga gtagacagag taggtagagt agacgggcgagtagatagag 2660

[0095] taggcgggcg agtagacagg gtagacgggg cgagtagata gagtaggagg gcgagtagacagggtagacg 2730

[0096] ggcgagtaga caggtagac gggcgagtag atagagtagg cggcgagta gacaggtagacggcgagt 2800

[0097] agatagta ggagggcgag tagacagggt agacggcaa gtagacaggt agacggcgagtagatagagag 2870

[0098] taggaggcga gtagacaggg tagacggggcg agtagatagg gtaggcggc gagtagacagagtagacggc 2940

[0099] gagtagacag tagacgggcg agtagacagg acgagagagc aaatattgat aatgaaggaatagggagaga 3010

[0100] tgggtagatg gatagagcaa atattgataa agaaggaata gggagaggag atgggtagatttagagca 3080

[0101] agtattgatg atgaaggtaa aggaaggga gatggataga tggataacga tgagggaaaaaagtagaga 3150

[0102] 3220

[0103] 3290

[0104] gaaggaggc ggtgagcgaa gaagagaag aagaaga gaagaaa aaaatcccgaagaagacaag 3360

[0105] aagacgtagg gaaaagaat aaaaagaaga taaaacgttt cattccaaga aaaaagattacagatatacg 3430

[0106] ttgaaccagc tgtcaaaacg accgcgctga ctagtttccg gtaaccaccc tcccccctccccctctccct 3500

[0107] atctccctcc tctctcccct cttctcttac tcactggact ctctctttct ctctctctctctgctaactg 3570

[0108] gactctctct ctctctctct ctctctcttt ttctcacttc tcacttactc actctcactgctctctctct 3640

[0109] ctctctctct ctctctctct ctctctctct ctctctctct ctctcttctc tcttctctctctctctctct 3710

[0110] ctctctctct ctcttactca ctggactttt ccccctctct gccttaatct ttcattttaatctttaatcc 3780

[0111] cctccctctt tgttccctaa attatctccc cttcttcccc cctctcccgt tatatctctaccccttctct 3850

[0112] cccctttctc tctctctctc tctctctctc tctctcctct ctctctctct ctctctctctctctctctct 3920

[0113] ctctctctct ctctctctct ctctctcttc tctctttctc tctctctttc attctcccctccctcccctc 3990

[0114] ccctctcttc cccttcccct ctaacacaag caaccccctt ccccttcccc ctttcaccccccccatttaa 4060

[0115] accaagccat gacctcccta cccccacccc ctccctaccc cccctctttc ctctctgaacgtgggctttg 4130

[0116] atcttgttgt ttcctttcca cattaacaac aacaacttag tcataaccgg cctttttgagtgctgccgga 4200

[0117] tttttgggtt cagtttgaca ttttgatttt tgctgtatat attttatttt attttatttttatattattt 4270

[0118] tattttcatt ttatttattt atatttgtgt cttttctctc tctctctctc tcgccgcctctgtcccttct 4340

[0119] ccaaaaattc ggttttcggt atttttgaaa atttgttttt tggcgatttc ttgtttgtagaggcactgtt 4410

[0120] atttctgtat tattattgtt ttttttatta tgattatatt atcattatta ttatttattattattat 4480

[0121] tattatatat tattatcatt attattatta ttaatcatta ttattatta ttattattangagttcgttg 4550

[0122] caaaaggtta ttaaagtgga aatgagttcg ttgtgcaagt aagactgtta ttcgaaattcaagatcacaa 4620

[0123] atcctatgaa ttttgaacat tcttgaacaa tgaatgaatt tgttcaaata ttccttgattttattggtat 4690

[0124] tgttcaggca gttctttagg aaaaaattta ttttaattac tttctaatcg catctttctttctttctctt 4760

[0125] tctctttctc ttctctttc tctttctctc tctctctct cctctctctt ccatctctctctctctc 4830

[0126] tctctttct ttctttcttc ttctttcttt tctctctct ctcttctctc tctctctctctctctctc 4900

[0127] tctctctct tctctctct tctctcttt cctctctct cctctctct cctctctctctctctct 4970

[0128] ctctctctc tctctcttg ctctctct ctctctct tttctgtcat tctgttttcagtgtagcaat 5040

[0129] ttctttcgtt ttctttacca tgtcgttttc taatttaatg aactaaatgg gagacttcttcaaactgaat 5110

[0130] cattatcatt taactgaatt attagtatta gtattattcg tattggtatt attgtagttattattgtatt 5180

[0131] atcttattat tatattatta ttttttattt tgttattatt gttattatat cgtcatcattatccttatta 5250

[0132] atgttgttct attattatta ttgctattat attatcatat tattattaca gcaatccttcttaccattat 5320

[0133] cattggtaat atcatcatat ttattaccgt tattgttgtt gcctatcttc tctttttcgcttattaatat 5390

[0134] atcatcatta ttattattat ttattattat attattatta ttatatatta ttatatattattatatatta 5460

[0135] ttatattatt attatatata cactagtact actatcatat cattattacc attactattaattgatgatt 5530

[0136] atcattattt tttattaata tcatttcatc ctccattatt accattatta ttaatttgatgattattatt 5600

[0137] atttttatta ttaatatcct catccttatc atcattacta ccactactat tactgttaacattactggta 5670

[0138] tcagtatttg attattactg atatcattag ataattaagt tttggtcagt tttgttttgttctattacta 5740

[0139] ttattattta ctattgttat tgtttattct aaggtgtagc attatttttg tttttatatcttatgacaat 5810

[0140] tattattttt atatatttct gctctgctct cctttcctcc cgctccattt ttattattcttgctatcacc 5880

[0141] ctattcctat tatcacgcat ctctttatac gtcttcgtta aatccctctc tatttccctcctttcttgct 5950

[0142] ccctcttgct tttttttatc ttttgttttt tttttttttt ttctgtctta ctcgtattctccctccccca 6020

[0143] tcatcttcta cccctccctc caacccctta cttttactct cccttatcat tactctcacacatctccatt 6090

[0144] aattctgttt gtcttctctc tctctccctc tcttctctac tccctctccc tctccctctccctctccctc 6160

[0145] tccctctccc tcctctctct ctctctctct cctctctccc caatctcact cccttaaccccttttattta 6230

[0146] aatccctctt ctctccccct ctctctccct tcctccctcc cccaccagcc cctttttacccctttattaa 6300

[0147] atccctctcc tcccctcttc tctccttcct ccatcccttc caccagccct tttataccctttattaaatc 6370

[0148] cctctccctc cttctctccc ttcctccctc cccctctccc ccctcctccc ctctctccccctccctcccc 6440

[0149] ttctccctcc ctccctcctc ctccctccct ccctccctcc tccctccctt ccttccaccagccccttttt 6510

[0150] acccctttat taattccccc ttctctccct tcctccagaa ccgccaagat gcccgtcgatctccgtgcca 6580

[0151] tcttcggcaa cgtcttgcac ctcttcaaga gccgcgccaa ccagatctgc gcggcgtcttgcgatgggct 6650

[0152] cgtcctgcgg atgcactaca ggtggacctt ctgcctgctc atggggaagg ttcctgaccgtgtggtactc 6720

[0153] tggtaagccc ccttcccccc ccttatttct gcctgctcat ggggatgttc ctgacgtgtggtactcatgg 6790

[0154] taagcccttc ccccttattt ctgcctgctc atgggatgtt cctgaccgtg gttactcatggtaagcccct 6860

[0155] tcccccccct tattctgctg ctcatgggat gttcctgacc gtgtgggtac tcatggtaagcccccttccc 6930

[0156] ccccttatt ctgcctgctc atggggatgt tcctgaccgt gtggtactca tggtaagcccccttcccccc 7000

[0157] ccttatttct gctgctcatg gggatgttcc tgaccgtgtg tactcatggt aaccccttcccccccccccc 7070

[0158] ttattctgc ctgctcatgg gatgttcctg accgtgtggt actcatggta agccccctcccccttatttc 7140

[0159] tgcctgctca tgggatgttc ctgaccgtgt ggtactcagg gaagcccccc tcccccccctttttagattt 7210

[0160] tttttggtt tatttgaatt taaattgata tttgttatgg tttcggcaaa gatttgtgtgtgtgtttatg 7280

[0161] 7350

[0162] atattatata tatatatata caacacacac acacacacac acacacac acacacacacacacacacac 7420

[0163] acaacacaca cacacatata gcattata tatatatata tatatatat ataatatactatatatat 7490

[0164] atatatataa tatatatata tatactatat atatatatgt atgttgtgtg tgttgtgtggtttcgcttt 7560

[0165] tgtctgttga taggaacgtt tctcacgctc cggtaatcaa gtagagtccc ttatagtttgttttgatccc 7630

[0166] cagtttgttt gtttgttggc agcgtttgtt tgcatttgtc tgtcggtttc tgtggcgtccgtggcttgcg 7700

[0167] ttgggttttc tgtctgtggt tggcgcgtcg ttgcctctct ctctattttt tttcatttttttctttcttc 7770

[0168] tgttcttatt ttttcgggtt atgggttgac tgggtggggt agttctttct tctgttcttttcttctctct 7840

[0169] cttcgttttg gttctcttct ctttttgtct gtctgtctgt ctgtctcgtc tctgtctcttctctctctct 7910

[0170] ctctctctct ctcctctctc tctctctctc tctctctctc tctctctctc tctctctctctctcctctcc 7980

[0171] tctctctctc tctcctcttc tcttcacttc tctcttttct tctctctctg tattctctgtctctctctct 8050

[0172] ttttcttctc cctctctctc tctctctctc tctctctctc tctctcctct ctctctctcttctctctctc 8120

[0173] tctctctctc tctctctctc tctctctctc cctctctccc tctcctctcc ctctccttctcccttctccc 8190

[0174] tttctccttc tcttctcctt tctcccttct cccttccct tctccctctc ccttctcccttattcacacc 8260

[0175] cagtcgctgc tacgagaaaa gtgcaagacg gtttgatctt tagcattctg gcgtcaccgaccgagacaat 8330

[0176] gagatcagat tatttctaag tgtgtcttga ttgctggctc ggaaatgttc gacaagccaaggcaagtcag 8400

[0177] ttgacaaccc acggcaagta agttcacaag tcaaagcaag atcgtttagg cagtcatgagagtcggttgg 8470

[0178] caagtcatgc tgaggtaatt tgcatcttta agagtcagtc aagtaagtca agtcgaaagtcattcaacga 8540

[0179] gttgagagaa gtcaagcaaa gagaagaaaa gcaagcaaaa aaggcaactc gagcgagatcgaagtcgagt 8610

[0180] caaggcgaag gcagataagt cgacaagcaa ggaataatca ataagacaag aaagtaaacaagatggaaag 8680

[0181] taatagagcg acgcgaaaca gctggagtct ggctggcgtt ccggtggctt tggggtggcgtgtcttttgc 8750

[0182] ctacgcgatc ttggtcggga aatgaggatc tatctctgtc tttctatctg tctgtttgtctttctctgta 8820

[0183] tgtctctacg tctttttcac tcactgtctg tctctatgtc tctctttttt ctctctcttctgtctttctg 8890

[0184] gtctctcttt tttctctctg tctatgtctt tctcgtctct ctttttttct cttctttctttctctgtctc 8960

[0185] tctgtctctc tctctctctc tctctctctc tcctctctct ctctctctct ctctctctctctctctctct 9030

[0186] ctcctctctc tctctctctc tctctctctc cctctccttc tctctctctc tctctctctctctctctctc 9100

[0187] tctctctctc ttctctctct cttctctctc tctctctctc tctctctctc tctctctctctctctctctc 9170

[0188] tctctctctc ttctctctct ctctcttctc tctctctctc tctctctctc tctctccgttgcgcaaactg 9240

[0189] ttgtcgtctt gtatagctaa atgaaatgtt tatttacgta aatgatagag atagatgcgtatatttggcg 9310

[0190] gctacgtgct tggtacgcga gagagagaga gagagagaga gagagagaga gagagagagagagagagaga 9380

[0191] gagagagaga gagagagaga gagagagaga gagagagaga gagagagagc gagagatgagcagagagaga 9450

[0192] gagagagcga gagagagaga gagagagaga gagagagaga gagagagaga gagagaacggaagggtacaa 9520

[0193] gcgaccacat gcgttattga tagctaaatc gccttgtagg atattcatca taatggtctacattcataag 9590

[0194] gatatgttga tgaatcatgc aaatggcgcg acaagcgatg cgaggagagg aagggaaggcgcgaggagag 9660

[0195] gacaggagcg caggggcgac aggagcgagg aaggggtgag gaggagagg aggagaaggaggaggatgg 9730

[0196] ggagagg aggggaggag gagagggtga ggagaaggt ggggaggag gagagggaggaggagagag 9800

[0197] gagggagg aggaggtgga ggagaggag aggggggga ggagggag gagagggaggaggaggaggagga 9870

[0198] aggagagag gagagaga gggggggagg aggagagga gagaggaga gagaggagaggagaggaga 9940

[0199] ggaggaggga ggagagggg aggagggga ggagaggga ggagaggagga ggagaggaggaggaggaggaggaggagg 10010

[0200] aggaggagga ggaggaggag gaggaggaag gaggaggaag gagggtgggg aggagaggaggaggaga 10080

[0201] ggaggaggag gaggggtgg ggaggaggag gagggaggag aggaggagga ggaggaggagggggggggtg 10150

[0202] ggaggaggag gaaggggtg gggaggggg aggaggagga ggaggaggaggaggaggaggaggaggagga 10220

[0203] ggagggag ggagggagga aggtaatggg gatgggagggaggaggaga ggaaaaaagaataagaataa 10290

[0204] gaagaagaag aagaagtttc taaaaggaaga tgagaagctt gaggaaaagg agctagaagatgtgaggagaag 10360

[0205] gaagagaaga agaagatagg aaaagtagag acagatggag gttaaggagagagaatccgggacgtagaa 10430

[0206] atcggagaaa cagcaaaaga aagaggataa ataaagaaag aaaaagagat gccttgatgaaagtggagac 10500

[0207] gaaggagact cctccgtttt ctttcagttc ttggtcccgt tttctatgga atctcaatggcgcttttgca 10570

[0208] cgtgtaaaag agtgtctttg aatgccttgt tacttgttgc ctccggcctc tcaagagtcaccgaggagga 10640

[0209] ggagggag gaggaggagg aggagaaggg gaggaggaag aggagagaa gggaggaggaagaggag 10710

[0210] aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaahhh

[0211] aaggaagt cgagggaa gaggggaga agtcgaggag gaaggggaag aaggaagaaggaggga 10850

[0212] agtaggaag gagggagg agggcgagtg ggagaaggaa gaaggaggag gaacaagaaatagg 10920

[0213] gaagagaa gacggcgagg agggaaca acaacgcgag ggagaagatg gagaggaagaaagagaaaa 10990

[0214] 11060

[0215] atcaaccgac cgatgcggag aatgacgaga atattgagtg acaataaaga agacgaagaagagaagaag 11130

[0216] aaaagaagaa gatagaaaaa agtaggaggg gcaatcgta gaccgagaag cgagggaaatgggagttgagg 11200

[0217] gtgtgtttat ggggggtgggg ggtgggggggg tgcttactga gaaggaggga gtggagagggggaataagaag 11270

[0218] agggaagggg aaggaagaagg ggcgagaaag agaaggaagga gtggttaagg gggagggtagagggagggaggggggg 11340

[0219] tgtaggggag gaagggaagg agaggtagag aattatttcc gaagaatgag agagagcgagaagagagaga 11410

[0220] gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gagagagagagagagagaag 11480

[0221] agagaaggtt tccgggaagg gcagaatacg agtaagaggg aaggaggggg ttaagggtaggggaggggga 11550

[0222] ggggagggct gggggtattg cggaagaggt tggtacatta agcatctcaa gacacacaagatacacgtgt 11620

[0223] tgggaccatc ttgtctttgt ctccagcgga gagtgcatct ctctctctct ctctcttctctctctctctc 11690

[0224] tctctctctc tctctctctc tctctctctc tctctctctc tctctctctc tctctctctctctctctctc 11760

[0225] tctctctctc tctctctctc tctactccat tttttcttcc atatatctca tctattttatcaacgttttt 11830

[0226] tctcaagtac cttttccgta agcaagtttc cccttcgtgc cccttaccag cgagtgattatcgttccaat 11900

[0227] aaggctcatt ttcgtcggtt gatttatgtc tccatgatca ttatcgtcga atagggaggtcaaaggtcag 11970

[0228] tcgctaatga aaacacatgt tcaggcggta acgcacattg ctcttgagaa atgcgttttctttgggggtg 12040

[0229] aagagtcttg aggggagact attgcaaatg cgagacaggc catttcgaag aggggaatttatcagttccg 12110

[0230] gttgttatga ttgtggttgt ggagtgttgg tgcttgttgg cttggtcgat tgtcggtgagttgatctctg 12180

[0231] ttttgatctg tgtattaggc tctgtctgtt tgtgtctgtg tctgtctctt tttctctttctctctctttc 12250

[0232] tttatctctc tctctctctc tctctctctc tctctctctc tctctctctc tctctctctctaactctctc 12320

[0233] attctcatct ctctctctct ctctctctct ctctctctct ctctgtctct ctctctctcccgttagtatc 12390

[0234] tccacctcta attccctccc cgccctttcc tctcccttct ccctccgcct cccttccgagaacgtatttt 12460

[0235] ctaagggcgt gccggtgcat agcgcagcca cctaattctc gccgtttact tccaggtattatcgtgacgt 12530

[0236] catcagctgc gtctcgcact tcaacgccga aacgcaagtc cgactcgact acatcaacatctgcctctcg 12600

[0237] tacccctacg tggaggacag cgggtcgagg cgctacctgc tcttctaccg gtggatctcgtggtcgctgc 12670

[0238] tggtgctcgc cttcatctac tacatcccga ggaagatgtc caagaacctg gacaactccaagtgcaagaa 12740

[0239] gctcctcgaa gacctggcgg ccaactcgag tcgctacgac caggcggagc gcgagctggtggaccgcgcc 12810

[0240] tccggctaca tcctgttcaa cctcaagacg cacaacggcc tctactggaa gttcctcgccgtcaacatcg 12880

[0241] tggcgctgtt cgtggacgtg ttcgccatgc agtacctgga cttcatcctg cagggccgcttcatccagta 12950

[0242] cggcttcaac tcgtacccgt tcaacaggga cccgcgcaac ttcaccgact acatgtcccagacgttcccg 13020

[0243] ccgttcgcgt cgtgcgagct ctccatcgag aaccagctga ccaaccggcg cacggagaagctcggttgcc 13090

[0244] acctcaccat catggagctc tacgagaagg tgttcctcgg cctgtggctc tggctcatcctgctcacgct 13160

[0245] caccacctgc tgctacctga tcttcctggg cctcatgtgg ctgccgcgga tgcagcagtacctcctgcgc 13230

[0246] acggccaagc cgggccacac caacagcaag atgggcagcg tcatcgagga cgcgatcaagaactgcaaga 13300

[0247] tcggcgacgt gtacctgctg taccgcgtga agcagcacct gagccacgcg cgcttctacgagctgctggc 13370

[0248] gcgcgtgtcg agccccaacc cgtacgccaa gccggtgccc ggcgcccccg agctcgagaagggcaagggc 13440

[0249] ggcccgccgg acatgggcaa gcagcagcaa atgcccgaca ccctccgcaa ccgcgcgcccgccatgccgc 13510

[0250] aggcgccgcc catgaaccag aacttctacc accagctgct ggccaacccc atgatggcccggccgccgca 13580

[0251] cccgcaggcc atgccgcccg gcccgcaggc catgccgcag ccgcagccca acaacaaggacgccaccaaa 13650

[0252] atcttagtcg actagtcacc gtggaggtct tgctagcccg tgggttgtgc cataccttagacagaaacaa 13720

[0253] aaaaaaaaaa tgaaaataga aaatagcact acttaaaact catagaaaac ttaaagacaaacgcaaggtg 13790

[0254] tcggtaacag caagaatcct agtcatcagg tgcagcgatt cgtccccaac gcctcccaagatttctctca 13860

[0255] tcttgccaac ggagtgtgca cctgaatttc tttatattgt gtgtgatatt taggtttcaatgatagtctt 13930

[0256] tcggaaaagg aaagacggaa tgacgtgtag gcttgctagc gacgcggctg ccaagacatagggctttgtg 14000

[0257] gatctgacga agggaggggc ccagccggta cccagttata taggcctata gtattgtttttttcaattcc 14070

[0258] tttttgttta ggttttaaag caatttttga gacttttcat tcgtcctatt taaatattctctcttaacga 14140

[0259] tgtatccagt ttaagtctcg ttttattttt ttttattcat attttgtgaa cctacagtatatgtacgcat 14210

[0260] ttgaagccag gtttgggaga agctttatat atatatatat 14250

[0261] Note: The underlined sequences in the text are, in order, the complementary sequences of the forward primer, the SNP site, and the reverse primer.

[0262] Example 2

[0263] In this embodiment, primers were designed for PCR amplification of the SNP sites obtained in Example 1, and the genotype of Litopenaeus vannamei tolerant to high ammonia nitrogen was obtained by sequencing. Subsequently, Litopenaeus vannamei with the high ammonia nitrogen tolerance trait was screened. The specific implementation method is as follows:

[0264] (S1) Obtaining Litopenaeus vannamei populations and high ammonia nitrogen treatment

[0265] A population of Litopenaeus vannamei was obtained using the same method as in Example 1, and another batch of 180 Litopenaeus vannamei were randomly selected and subjected to high ammonia nitrogen stress treatment using the same method as in Example 1 for large-scale population verification.

[0266] (S2) Extraction of Litopenaeus vannamei DNA

[0267] Gill tissues were collected from 100 shrimp exhibiting extreme phenotypes (180 Litopenaeus vannamei shrimp were subjected to ammonia nitrogen stress, and the 50 shrimp that died fastest and the 50 that died last under ammonia nitrogen stress were selected as extreme phenotypes). DNA was extracted from the gill tissues using the MolPure Cell / Tissue DNA Kit (Yeasen). The specific procedures are as follows:

[0268] (1) Remove the shrimp individuals that are lying on their side and do not respond to touch; take the gill tissue of each shrimp and freeze it in liquid nitrogen for DNA extraction;

[0269] (2) Add 400 μL of lysis buffer LB to a 1.5 mL sterile centrifuge tube, then add the gill tissue and two grinding beads to it, and grind it in a high-throughput tissue homogenizer. The program is set to 70 Hz and 60 s. After grinding, add 10 μL of Proteinase K to the centrifuge tube and vortex to mix thoroughly.

[0270] (3) Place the sample in a 55°C constant temperature water bath for 10 min to 20 min, and shake the sample 2 to 3 times during the period, each time for 15 s, until the solution is clear and transparent;

[0271] (4) After the centrifuge tubes are brought to room temperature in a water bath, shake to mix the system. Add 300 μL of protein removal solution PL and 300 μL of binding solution BD in sequence, and shake vigorously to ensure thorough mixing;

[0272] (5) Place the adsorption column T1 in a 2 mL collection tube, add 200 μL of buffer AC, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid;

[0273] (6) Centrifuge the sample in (4) at 12,000 rpm for 5 min. After centrifugation, the solution will separate into layers. The DNA is present in the lower aqueous phase. Carefully aspirate the lower solution into (5) using a pipette, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

[0274] (7) Place the adsorption column T1 in the collection tube, add 500 μL of washing buffer W, centrifuge at 12,000 rpm for 30 s, and discard the filtrate. Repeat this step once;

[0275] (8) Place the adsorption column T1 in the collection tube and centrifuge at 12,000 rpm for 2 min to completely remove the rinse solution residue;

[0276] (9) Place the adsorption column T1 into a new 1.5 mL centrifuge tube, add 50 μL of elution buffer to the center of the adsorption column, let stand at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min. Collect the filtrate, which is the required DNA solution;

[0277] (10) After mixing 1 μL of DNA solution with loading buffer, the integrity of DNA was detected by 1% agarose gel electrophoresis. Then, its concentration was detected by Biochrom SimpliNano ultra-micro spectrophotometer. After making the corresponding records, the sample was temporarily stored in an ultra-low temperature freezer at -80°C.

[0278] (S3) PCR amplification of the target fragment

[0279] Primers were designed using Primer Premier 5 software based on the LvInx2 gene nucleotide sequence of Litopenaeus vannamei (as shown in SEQ ID NO: 1) targeting the SNP site 680A>G. The primers included a forward primer LvInx2-F and a reverse primer LvInx2-R, as shown below: Forward primer LvInx2-F: 5'-gctacactgcctggttgtct-3' (as shown in SEQ ID NO: 2); Reverse primer LvInx2-R: 5'-gggaaaacaaggccgcaaat-3' (as shown in SEQ ID NO: 3).

[0280] Using DNA extracted from the gill tissue of Litopenaeus vannamei in step (S2) as a template, the target fragment (440 bp) was amplified by PCR using the following reaction system and program. The PCR reaction system was 20 μL, including 10 μL of 2×MagicTaq PCR Super(+dye), 1 μL each of 10 mM forward and reverse primers, 1 μL of 40 ng / μL DNA template, and 7 μL of ddH2O. The PCR reaction program consisted of 32 cycles. The pre-denaturation was performed at 95℃ for 5 min before each cycle. Each cycle included denaturation at 94℃ for 30 sec, annealing at 57℃ for 30 sec, extension at 72℃ for 30 sec, and a final extension at 72℃ for 10 min after each cycle.

[0281] The target fragment (as shown in SEQ ID NO: 4) was amplified by specific primer PCR as follows:

[0282] GCTACACTGCCTGGTTGTCTCCCAGGCCAAGAGCAGATAATAGTCGACCTAAGGACTCTTATCAGAGGTGAGTGAT630TCCCAGACGCGGTTCCTTTCTTCCCATTAGCGTTTCGGCGCCAGTGTGAA[A(680A>G)]CAGATAGGAGTGATGTTAGCATTGTTTTTTTATGCTTTATGGACGCGAGTGTGTTGTGCTGGAATGAGAAGTAGGGAATGGATAGAA TTAATGATGCTCTTTGGCCAAGAACAATGGCTTCCCAGTTTTTCCTCCCCCAGTCTCGGTTAATTCGGTAGGAAATAGGTAGGCCTGGTGACGTCCATTCCTTTGAATTTGGGAGGACATACTTGCAGAAGCGATCAGCTAGTTTACCCTTTGTTCTGCGGGATCGACCCTTGCATTGTTCAGGGAATACTCGCGTAAATCGGGGTGT ATTTGCGGCCTTGT TTTCCC (Shown SEQ ID NO: 4);

[0283] The underlined part indicates the location of the complementary sequence of the forward and reverse primers, and the square brackets indicate the SNP sites to be detected.

[0284] (S4) Sequencing the PCR amplification products to determine the genotype of the SNP loci.

[0285] The PCR amplification products of 100 Litopenaeus vannamei individuals were sequenced at Beijing Qingke Biotechnology Co., Ltd. using direct sequencing (Sanger sequencing). Genotyping analysis was performed on the sequencing peaks using SnapGene software. A double peak at the SNP site indicated a heterozygous genotype, while a single peak indicated a homozygous genotype. This determined the 680A>G genotype. The results are as follows: Figure 1 As shown.

[0286] (S5) Correlation analysis of SNP locus genotypes and high ammonia nitrogen tolerance in Litopenaeus vannamei

[0287] One-way ANOVA in a general linear model using SPSS 26 was used to analyze the correlation between SNP genotypes and high ammonia nitrogen tolerance quantitative traits. For SNPs with significant differences, Ducan's method was used for multiple comparison analysis. The results are shown in Table 1 below:

[0288] Table 1. Correlation between SNP sites in the LvInx2 gene of Litopenaeus vannamei and high ammonia nitrogen tolerance trait.

[0289]

[0290] The p-value represents the result of one-way ANOVA; the superscript letters for high ammonia nitrogen tolerance time indicate the results of Ducan's multiple comparison analysis between genotypes, with different letters indicating significant differences and the presence of the same letter indicating no significant differences.

[0291] The results in Table 1 show that there were significant differences (P<0.01) between the three genotypes at the SNP site (680A>G) on the LvInx2 gene of Litopenaeus vannamei and the high ammonia nitrogen tolerance trait, indicating that this SNP site is significantly associated with high ammonia nitrogen tolerance. Among them, Litopenaeus vannamei individuals with genotype AA had the longest survival time in high ammonia nitrogen tolerance (i.e., AA is the dominant genotype), while individuals with genotype GG had the shortest survival time (i.e., GG is the inferior genotype), and there was a significant difference between the two. This indicates that in the process of breeding Litopenaeus vannamei populations with high ammonia nitrogen tolerance, individuals with SNP genotype AA should be preferentially selected as parents for breeding Litopenaeus vannamei with high ammonia nitrogen tolerance.

[0292] When the SNP molecular marker described in this invention is applied to the breeding process of Litopenaeus vannamei for high ammonia nitrogen tolerance, specifically during the breeding process, genomic DNA of the Litopenaeus vannamei to be tested is extracted. Using primers designed in this invention, the genomic DNA of the Litopenaeus vannamei to be tested is amplified by PCR to obtain PCR amplification products. The PCR amplification products are then sequenced. Based on the sequencing results, the genotype of the SNP locus is detected in the candidate population for Litopenaeus vannamei breeding. Combined with the genotypes of other loci related to growth traits, disease resistance, and stress resistance, individuals with the SNP genotype AA described in this invention are preferentially selected as parents for breeding Litopenaeus vannamei with high ammonia nitrogen tolerance.

[0293] Therefore, the reagents, primers, or kits used to detect the SNP sub-labels can be applied in the identification and selective breeding of high ammonia nitrogen traits in Litopenaeus vannamei.

[0294] Furthermore, the application of reagents for detecting the SNP molecular markers, the primers or kits described therein, in evaluating the tolerance of Litopenaeus vannamei to high ammonia nitrogen levels is also within the scope of protection of this invention.

[0295] The above embodiments are only used to illustrate the present invention, and the scope of protection of the present invention is not limited to the above embodiments. Those skilled in the art can achieve the purpose of the present invention based on the above disclosure. Any improvements and modifications made based on the concept of the present invention fall within the scope of protection of the present invention, and the specific scope of protection is determined by the claims.

Claims

1. An amplification method for Litopenaeus vannamei LvInx2 Primers for SNP molecular markers related to the gene tolerance to high ammonia nitrogen trait, characterized in that, The primers include forward primers. LvInx2- F and reverse primer LvInx2 -R, the forward primer LvInx2- The nucleotide sequence of F is shown in SEQ ID NO: 2, and the reverse primer LvInx2 The nucleotide sequence of -R is shown in SEQ ID NO: 3; the SNP molecular marker is located in Litopenaeus vannamei. LvInx2 The gene has the nucleotide sequence shown in SEQ ID NO: 1, wherein the nucleotide sequence shown in SEQ ID NO: 1 has a base of A or G at position 680 from the 5' end.

2. A detection method for Litopenaeus vannamei LvInx2 A kit for SNP molecular markers associated with the trait of high ammonia nitrogen tolerance, characterized in that, The kit includes the primers as described in claim 1.

3. A method for detecting the high ammonia nitrogen tolerance trait of Litopenaeus vannamei, characterized in that, Includes the following steps: (S1) Extract genomic DNA from the gill tissue of Litopenaeus vannamei; (S2) Using the primers described in claim 1, the genomic DNA of the gill tissue of Litopenaeus vannamei described in step (S1) is amplified by PCR to obtain the amplification product; (S3) Sequencing the amplification products described in step (S2), and determining the genotype of the SNP molecular marker based on the sequencing results; (S4) The high ammonia nitrogen tolerance trait of Litopenaeus vannamei is determined by the genotype of the SNP molecular marker described in step (S3); the high ammonia nitrogen tolerance trait of individuals with the AA genotype of the SNP molecular marker is significantly higher than that of individuals with the GG genotype.

4. The method for detecting the high ammonia nitrogen tolerance trait of Litopenaeus vannamei according to claim 3, characterized in that, The PCR reaction system used in step (S2) for PCR amplification is 20 μL, including: 10 μL of 2×MagicTaq PCR Super, 1 μL each of 10 mM forward and reverse primers, 1 μL of 40 ng / μL DNA template, and 7 μL of ddH2O.

5. The method for detecting the high ammonia nitrogen tolerance trait of Litopenaeus vannamei according to claim 3, characterized in that, In step (S2), the PCR amplification process used a total of 32 cycles. The pre-denaturation was performed at 95°C for 5 min before each cycle. Each cycle included denaturation at 95°C for 30 sec, annealing at 57°C for 30 sec, extension at 72°C for 30 sec, and extension at 72°C for 10 min after each cycle.

6. Testing Litopenaeus vannamei LvInx2 The application of reagents, primers as described in claim 1, or kits as described in claim 2, for identifying or selecting Litopenaeus vannamei varieties with high ammonia nitrogen tolerance traits; wherein the SNP molecular marker is located in Litopenaeus vannamei. LvInx2 The gene has the nucleotide sequence shown in SEQ ID NO: 1, and the nucleotide sequence shown in SEQ ID NO: 1 has a base of A or G at position 680 from the 5' end; individuals with the AA genotype of the SNP molecular marker have significantly higher tolerance to high ammonia nitrogen than individuals with the GG genotype.

7. The application of the detection method according to any one of claims 3-5 in the identification or selection of Litopenaeus vannamei varieties with high ammonia nitrogen tolerance.

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