Vannamei white shrimp anti-vibrio parahaemolyticus infection trait related snp marker and application thereof

Abstract

The application discloses a SNP marker related to the Vibrio parahaemolyticus infection resistance of Litopenaeus vannamei and application thereof, and belongs to the technical field of aquatic breeding organisms. The application provides a SNP molecular marker related to the Vibrio parahaemolyticus infection resistance of Litopenaeus vannamei, which is characterized by being located at a 416bp site of a C-type lectin gene sequence coding region (SEQ ID NO. 2) of the Litopenaeus vannamei, and the mutation type is T / T homozygous type, T / A heterozygous type and A / A homozygous type; when the variation site is A / A, the resistance of the Litopenaeus vannamei to the Vibrio parahaemolyticus is stronger. Meanwhile, the application provides SNP site amplification primers, and establishes a molecular marker assisted selection system for the Vibrio parahaemolyticus resistance of the Litopenaeus vannamei, so that the breeding process of the Vibrio parahaemolyticus resistance of the Litopenaeus vannamei can be accelerated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of aquatic breeding biotechnology, and particularly relates to a SNP marker related to the anti-Vibrio parahaemolyticus infection trait of Litopenaeus vannamei and application thereof. BACKGROUND

[0002] Litopenaeus vannamei, commonly known as South American white shrimp, is the world's first farmed shrimp variety. In China, the yield of Litopenaeus vannamei farming in 2020 was more than 1.86 million tons, accounting for 86% of the annual output of shrimp in China. The production of shrimp fry was more than 1,500 billion, and the market demand for good varieties was huge. Except for Beijing, Jilin, Tibet and Qinghai, etc. There is no farming in other provinces and cities. Starting from the germplasm level, in-depth exploration and utilization of Litopenaeus vannamei's antibacterial germplasm is of great significance to the healthy development of Litopenaeus vannamei farming.

[0003] In recent years, the outbreak of Litopenaeus vannamei disease is more frequent, and Vibrio disease is the most common and most harmful disease in shrimp farming. The "acute hepatopancreas necrosis syndrome (AHPND)" caused by Vibrio parahaemolyticus infection causes great loss to Litopenaeus vannamei farming. The AHPND pathogenic plasmid has the ability of vertical and horizontal transmission, which makes the AHPND of Litopenaeus vannamei highly prevalent and highly outbreak. Litopenaeus vannamei AHPND develops rapidly from infection to disease outbreak, with a mortality rate of 100% within 1-3 days, causing great loss to farming production.

[0004] C-type lectin can recognize invading pathogenic microorganisms and trigger immune response in the innate immune system of invertebrates. Lectin can bind, agglutinate and inhibit the growth of pathogenic microorganisms; at the same time, lectin can also bind to receptors on phagocytes to enhance their phagocytosis. Studies have shown that C-type lectin is an important immune-related functional gene in Litopenaeus vannamei. SUMMARY

[0005] To solve the above problems, the present application provides a SNP marker related to the anti-Vibrio parahaemolyticus infection trait of Litopenaeus vannamei, amplification primers and application thereof, which is used for the assisted breeding of Litopenaeus vannamei antibacterial trait excellent varieties, thereby speeding up the breeding process of Litopenaeus vannamei antibacterial trait excellent varieties.

[0006] In the present application, the genomes of Vibrio parahaemolyticus resistant and susceptible shrimp are used as templates, and the genotypes of SNP sites on the C-type lectin gene of Litopenaeus vannamei are detected by PCR amplification and sequencing. The SNP marker related to the anti-Vibrio parahaemolyticus trait of Litopenaeus vannamei is determined by association analysis, and the SNP site amplification primer is provided to establish a molecular marker assisted breeding system for Litopenaeus vannamei anti-Vibrio parahaemolyticus trait excellent varieties.

[0007] The first object of the present application is to provide a SNP molecular marker related to the Vibrio parahaemolyticus resistance trait of Litopenaeus vannamei, which is located at the 416bp site of the coding region of the Litopenaeus vannamei C-type lectin gene sequence, the base is T or A, the mutation type is T / T homozygous, T / A heterozygous and A / A homozygous, and the nucleotide sequence of the coding region of the Litopenaeus vannamei C-type lectin gene sequence is shown in SEQ ID NO. 2.

[0008] The second object of the present application is to provide a detection primer of a SNP marker related to the Vibrio parahaemolyticus resistance trait of Litopenaeus vannamei, comprising the following primers:

[0009] LvCTL-SNP-F: 5'-GTGAGGTCTTGCGCAAATATC-3',

[0010] LvCTL-SNP-R: 5'-CCTCGTGTATCTGAATAAGGGC-3'.

[0011] The third object of the present application is to provide a kit containing the above detection primers.

[0012] The fourth object of the present application is to provide the use of the above SNP molecular marker, detection primer or kit in identifying or breeding Vibrio parahaemolyticus-resistant Litopenaeus vannamei.

[0013] Preferably, the breeding of Vibrio parahaemolyticus-resistant Litopenaeus vannamei comprises the following steps:

[0014] a. extracting genomic DNA from the swimmer of Litopenaeus vannamei parent shrimp;

[0015] b. PCR amplifying the DNA of individual Litopenaeus vannamei using LvCTL-SNP-F and LvCTL-SNP-R primers;

[0016] c. sequencing the amplification product obtained in step b to determine the genotype of the SNP marker of claim 1, and selecting A / A homozygous female and male shrimp as parents for breeding of Vibrio parahaemolyticus-resistant Litopenaeus vannamei.

[0017] More preferably, the PCR amplification, the PCR system and its final concentration composition are as follows: PCR Buffer 1x, high-fidelity PCR enzyme 0.02 U / μL, dNTP Mixture 200 μM each, MgCl2 2mM, LvCTL-SNP-F 0.2 μM, LvCTL-SNP-R 0.2 μM, shrimp genomic DNA 0.25 ng / μL, and the rest is made up to 25 μL with sterile double distilled water.

[0018] More preferably, the PCR amplification has the following reaction procedure: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 30 seconds, 35 cycles in total; and 72℃ re-extension for 6 minutes.

[0019] Preferably, the amplification product obtained in step b is sequenced using the primers LvCTL-SNP-F or LvCTL-SNP-R.

[0020] Compared with the prior art, the present application has the beneficial effects that the present application discloses a credible SNP molecular marker, when the variation site is A / A, the resistance ability of Litopenaeus vannamei to Vibrio parahaemolyticus is stronger; the present application further provides a primer combination and a breeding method for detecting the SNP site, in the process of breeding of the Litopenaeus vannamei Vibrio-resistant variety, the parent can be screened through the method, the breeding process can be significantly accelerated, and the method has important guiding significance for improving the breeding of the Litopenaeus vannamei disease-resistant excellent variety, the method is high in efficiency, simple in operation, and stable in genotype of the breeding individual and does not cause genetic differentiation. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Fig. 1 is a peak chart of the T / T homozygous type of the SNP site of the Litopenaeus vannamei C-type lectin gene. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and beneficial technical effects of the present application clearer, the present application is further described in detail below in combination with examples. It should be understood that the examples described in the present specification are only for explaining the present application, and are not intended to limit the present application, and the parameters, ratios and the like of the examples can be selected as appropriate, and have no substantial influence on the results. In the examples, the reagents and method steps are conventional in the art unless otherwise specified.

[0023] Example 1 Determination of SNP molecular marker

[0024] Thirty-six Litopenaeus vannamei were collected from a shrimp farm in Zhanjiang, and genomic DNA was extracted, and then amplified using the primers LvCTL-SNP-F and LvCTL-SNP-R, and the specific method was as follows:

[0025] Amplification primer:

[0026] LvCTL-SNP-F: 5'-GTGAGGTCTTGCGCAAATATC-3',

[0027] LvCTL-SNP-R: 5'-CCTCGTGTATCTGAATAAGGGC-3';

[0028] Amplification system:

[0029]

[0030] PCR amplification reaction program is preferably: 95℃ pre-denaturation 5 minutes; 95℃ denaturation 30 seconds, 60℃ annealing 30 seconds, 72℃ extension 30 seconds, a total of 35 cycles; 72℃ extension for 10 minutes again. The PCR amplification product is sequenced using LvCTL-SNP-F or LvCTL-SNP-R primer. The nucleotide sequence of the amplification product is shown in SEQ ID NO. 3.

[0031] It is found that the Litopenaeus vannamei bred in the place contains three mutation types at the 416bp position of the coding region of the lectin gene, which are T / T homozygous type, A / A homozygous type and T / A heterozygous type, and the sequencing peak chart is shown in Figure 1 When the site is T, the 139th amino acid of the coding protein sequence is phenylalanine (F); when the site is A, the 139th amino acid of the coding protein sequence is tyrosine (Y).

[0032] The cDNA sequence of the Litopenaeus vannamei C-type lectin gene (SEQ ID NO. 1) is shown as follows, wherein the underlined sequence is the coding region sequence (SEQ ID NO. 2), and the bold and capital letter T is the mutation site:

[0033]

[0034] Example 2 Detection and typing of SNP molecular marker

[0035] 200 Litopenaeus vannamei with body length of 5-6cm from a shrimp farm in Zhanjiang are used for Vibrio parahaemolyticus challenge test. The Vibrio parahaemolyticus used in the challenge test is isolated from the liver and pancreas of Litopenaeus vannamei with acute hepatopancreas necrosis syndrome by the South China Sea Institute of Chinese Academy of Sciences, and is stored in 25% glycerol at -80℃. The Vibrio parahaemolyticus is streaked on 2% TSB solid medium (1L distilled water added with: tryptone 17g; soybean peptone 3g; D-glucose 2.5g; NaCl 5.0g; K2HPO4 2.5g; pH 7.3 ±0.2), and is cultured at 30℃ overnight; a single colony is selected and inoculated in 2% TSB liquid medium, and is cultured at 30℃ with shaking overnight; the bacterial liquid is centrifuged at 3000rpm for 5 minutes to collect the amplified Vibrio parahaemolyticus; the bacterial liquid is resuspended with pre-cooled PBS buffer, and is centrifuged, which is repeated for 3 times; the concentration of the resuspension is determined by spectrophotometer at 600nm (OD 600 ).

[0036] The Litopenaeus vannamei is placed in a 1m 3 black large plastic bucket, and is temporarily cultured for 2 weeks at 30‰ salinity and 30℃. The Vibrio parahaemolyticus liquid is poured into the black large plastic bucket of the temporarily cultured Litopenaeus vannamei, and the concentration is adjusted to 2.86x105 cfu / mL. The mortality of Litopenaeus vannamei was observed every 2h during the challenge, and the dead individuals were collected and immersed in 95% alcohol in time, and observed for 1 week. The individuals died within 2 days (48h) before the challenge were defined as the susceptible population of Vibrio parahaemolyticus, and the individuals still alive after 7 days of challenge were defined as the resistant population. 36 individuals were randomly selected from the resistant and susceptible populations of Litopenaeus vannamei, respectively, to extract genomic DNA, and the resistant and susceptible populations of Vibrio parahaemolyticus were detected by PCR using the primers LvCTL-SNP-F and LvCTL-SNP-R described in Example 1, and the C-type lectin SNP marker was typed, and the results are shown in the following table:

[0037]

[0038] As can be seen from the above table, the genotypes of C-type lectin of most resistant groups of Litopenaeus vannamei are A / A, indicating that the individuals with A / A genotype have stronger ability to resist Vibrio parahaemolyticus infection; and the genotypes of C-type lectin of the susceptible group are mostly T / T, indicating that the individuals with T / T genotype are more susceptible to Vibrio parahaemolyticus infection.

[0039] Example 3 Application of the SNP molecular marker in the breeding of Litopenaeus vannamei against Vibrio parahaemolyticus

[0040] The swimmerets of Litopenaeus vannamei from the same batch of ripening parent shrimp were taken from a Litopenaeus vannamei seedling farm in Zhanjiang, and genomic DNA was extracted, and the genotypes of the C-type lectin SNP marker of the parent shrimp were typed according to the method of Example 1. One pair of parent shrimp with T / T homozygous genotype and one pair of parent shrimp with A / A homozygous genotype were selected for breeding. When the full-sibling juvenile shrimp of the F1 generation grew to 3 cm in length, 100 individuals from each family were subjected to Vibrio parahaemolyticus challenge test (methods and steps same as those of Example 2). Among them, 68 individuals (68%) of the juvenile shrimp produced by the parent shrimp with A / A homozygous genotype survived; and 29 individuals (29%) of the juvenile shrimp produced by the parent shrimp with T / T homozygous genotype survived. It can be seen that the individuals of Litopenaeus vannamei with A / A genotype at the SNP site have better ability to resist Vibrio parahaemolyticus infection, and the trait can be stably inherited in the offspring. Therefore, the SNP marker disclosed in the present application can be used as a marker for breeding Litopenaeus vannamei against Vibrio parahaemolyticus, and the individuals of Litopenaeus vannamei with A / A homozygous genotype can be selected as candidate parents in selective breeding. SEQUENCE LISTING <110> South China Sea Institute of Oceanology, Chinese Academy of Sciences <120> A SNP marker related to Vibrio parahaemolyticus infection resistance trait of Litopenaeus vannamei and application thereof <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 618 <212> DNA <213> Litopenaeus vannamei <400> 1 ggtagcgcat aacaactcgc tggccaatat aaagcacagc attgctcttg aaggcaacac 60 aaacgcgcaa aatgatgttc ttcgtgctcc tgctgtcctt cctgccactg ctgacaggct 120 ctctactccc atctcaatca gccaccacac agataacttg tgatgaaggt tttcacaaca 180 tttacgaccg ctgcgtccag ttccgcacgc aggaagtctc gtggcacgag gggaagaacg 240 agtgctttaa catgggggcc gaactagcca aggtcgatga cgcaaacttc atgtactacc 300 tcgtcaagtt catcagaaat aatgggctcg acggtcacaa ttactggatc ggcgcctcag 360 acgaaggcca cgaaggcgac ttcaggtgga cggacgggac ggaggtcaag atggggacgc 420 cgttctgggg cgacagcgaa gaccaggttc aggagcccga cggtggtacc aaccagaact 480 gcgtgttcat ggccagaggc gatcacttct tcttttttga ttatgattgt agccctccac 540 aagccattat ctgtgagaaa tttatgtagt tttagtagag gaataaaaaa ataaattctt 600 aatacaagaa caccgata 618 <210> 2 <211> 498 <212> DNA <213> Litopenaeus vannamei <400> 2 atgatgttct tcgtgctcct gctgtccttc ctgccactgc tgacaggctc tctactccca 60 tctcaatcag ccaccacaca gataacttgt gatgaaggtt ttcacaacat ttacgaccgc 120 tgcgtccagt tccgcacgca ggaagtctcg tggcacgagg ggaagaacga gtgctttaac 180 atgggggccg aactagccaa ggtcgatgac gcaaacttca tgtactacct cgtcaagttc 240 atcagaaata atgggctcga cggtcacaat tactggatcg gcgcctcaga cgaaggccac 300 gaaggcgact tcaggtggac ggacgggacg gaggtcaaga tggggacgcc gttctggggc 360 gacagcgaag accaggttca ggagcccgac ggtggtacca accagaactg cgtgttcatg 420 gccagaggcg atcacttctt cttttttgat tatgattgta gccctccaca agccattatc 480 tgtgagaaat ttatgtag 498 <210> 3 <211> 330 <212> DNA <213> Litopenaeus vannamei <400> 3 gtgaggtctt gcgcaaatat cggtgttctt gtattaagaa tttatttttt tattcctcta 60 ctaaaactac ataaatttct cacagataat ggcttgtgga gggctacaat cataatcaaa 120 aaagaagaag tgatcgcctc tggccatgaa cacgcagttc tggttggtac caccgtcggg 180 ctcctgaacc tggtcttcgc tgtcgcccca gaacggcgtc cccatcttga cctccgtccc 240 gtccgtccac ctgaagtcgc cttcgtggcc ttcgtctgag gcgccgatcc agtaattgtg 300 accgtcgagc ccttattcag atacacgagg 330

Claims

1. A SNP molecular marker associated with the anti-Vibrio parahaemolyticus infection trait in Litopenaeus vannamei, characterized by, The nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO. 2, and the mutation type at the 416 bp site of SEQ ID NO. 2 is T / T homozygote, T / A heterozygote and A / A homozygote.

2. Use of the SNP molecular marker of claim 1, or a detection primer for detecting the SNP molecular marker or a kit containing the detection primer in the preparation of identifying or selecting Litopenaeus vannamei products resistant to Vibrio parahaemolyticus, wherein individuals with A / A genotype are more resistant to Vibrio parahaemolyticus infection, and individuals with T / T genotype are more susceptible to Vibrio parahaemolyticus infection. The detection primer comprises the following primers: LvCTL-SNP-F: 5'-GTGAGGTCTTGCGCAAATATC-3', LvCTL-SNP-R: 5'-CCTCGTGTATCTGAATAAGGGC-3'.

3. Use according to claim 2, characterized in that, The selection of Litopenaeus vannamei resistant to Vibrio parahaemolyticus comprises the following steps: a. Extracting genomic DNA from the swimmerets of Litopenaeus vannamei parent shrimp; b. PCR amplifying the DNA of individual Litopenaeus vannamei using the primers LvCTL-SNP-F and LvCTL-SNP-R; c. Sequencing the amplification product obtained in step b to determine the genotype of the SNP marker of claim 1, and selecting female and male shrimp with A / A homozygote as parents for the selection of Litopenaeus vannamei resistant to Vibrio parahaemolyticus.

4. Use according to claim 3, characterized in that, The PCR amplification has the following PCR system and final concentration composition: PCR Buffer 1x, high-fidelity PCR enzyme 0.02 U / μL, dNTP Mixture 200 μM each, MgCl2 2 mM, LvCTL-SNP-F 0.2 μM, LvCTL-SNP-R 0.2 μM, shrimp genomic DNA 0.25 ng / μL, and the rest is made up to 25 μL with sterile double distilled water.

5. Use according to claim 3, characterized in that, The reaction program of the PCR amplification is as follows: pre-denaturation at 95℃ for 5 minutes; denaturation at 95℃ for 30 seconds, annealing at 60℃ for 30 seconds, extension at 72℃ for 30 seconds, for a total of 35 cycles; and further extension at 72℃ for 6 minutes.

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