MNP marker site for identifying fast-growing population of vannamei shrimp, primer set and application thereof

Abstract

The application provides a MNP marker site for identifying a fast-growing Litopenaeus vannamei population, a primer set and application thereof, and belongs to the technical field of molecular genetics of aquatic animals. The MNP marker site, the corresponding primer set and the kit thereof can screen the fast-growing Litopenaeus vannamei population, and it is proved that the MNP marker site can realize high-throughput analysis, avoid a large number of non-existent false genotypes, reduce detection cost and analysis complexity, and the accuracy of identifying the fast-growing Litopenaeus vannamei population is as high as 100%. The MNP marker site and the application thereof provided by the application have reference significance for the field related to molecular genetics and genotype analysis of aquatic animals.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics technology for aquatic animals, specifically relating to an MNP marker site, primer set, and its application for identifying fast-growing Litopenaeus vannamei populations. Background Technology

[0002] With the rapid expansion of the global Litopenaeus vannamei aquaculture industry and the cross-border flow of commercial germplasm, commercial strains bred in different countries exhibit significant differences in genetic background, phenotypic expression, and adaptability. For example, strains such as the US SIS (Shrimp Improvement System), Thailand CP (CP), and Thailand TOP (Top Fung) are characterized by rapid growth; strains such as the US API (American Shrimp Company), US Primo, and Mexico Blue Genetics exhibit high disease resistance; and the US Kona Bay strain combines both rapid growth and high disease resistance. Without accurate identification methods, problems such as germplasm mixing, breed infringement, and inbreeding depression can easily arise, directly impacting aquaculture efficiency and the sustainable development of the industry. Genetic identification of foreign commercial populations using specific molecular markers can ensure the purity of introduced strains, avoid production risks caused by germplasm mixing, and thus protect the rights of breeders and the safety of the breeding industry.

[0003] The molecular markers currently used for identification of plant and animal germplasm mainly include SSR and SNP, both of which have certain limitations. SSR markers rely on capillary electrophoresis or polyacrylamide gel electrophoresis, and the number of sites detected in a single test is limited, making it difficult to achieve high-throughput analysis. The manual operation steps are numerous and prone to human error. When polymerase amplifies simple repetitive sequences in SSR markers, there is a "slippage" phenomenon, which leads to the generation of a large number of non-existent erroneous genotypes. A single SNP only provides biallelic information, and a large number of sites (usually hundreds to thousands) need to be combined to achieve high-precision identification, which increases the detection cost and analysis complexity. Mainstream SNP genotyping technologies (such as microarrays and sequencing) require expensive instruments and bioinformatics analysis capabilities, which are not friendly to small-scale laboratories. In this regard, polynucleotide polymorphism (MNP) markers are a more ideal identification method with the following advantages: (1) rich alleles and high resolution. MNP markers have up to 2 n (1) Allele genotype (where n is the number of SNP markers in the MNP); (2) MNP marker typing results standard, and results under different experimental conditions can be accurately compared; (3) High efficiency. A single PCR reaction can amplify several to dozens of SNP sites. Developing population-specific MNP markers can enable the identification of commercial populations of Litopenaeus vannamei with important fast growth characteristics abroad. Summary of the Invention

[0004] The purpose of this invention is to provide an MNP marker locus, primer set, and application for identifying fast-growing Litopenaeus vannamei populations. The MNP marker locus screened in this invention can screen for fast-growing Litopenaeus vannamei populations. The MNP marker locus enables high-throughput analysis, avoids generating a large number of non-existent erroneous genotypes, reduces detection costs and analytical complexity, and has reference value for the fields of molecular genetics and genotype analysis of aquatic animals.

[0005] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution:

[0006] This invention provides an MNP marker locus for identifying fast-growing Litopenaeus vannamei populations, the location of which is shown below:

[0007] ;

[0008] ;

[0009] ;

[0010] ;

[0011] ;

[0012] ;

[0013] .

[0014] The present invention also provides a primer set for identifying fast-growing Litopenaeus vannamei populations, the primer set being used to detect the MNP marker sites, and the sequences of the primer set being shown in SEQ ID No. 1-SEQ ID No. 56.

[0015] Furthermore, the primer set includes primer pairs 1 to 28, each primer pair including a forward primer and a reverse primer, the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the 28th primer pair and the reverse primer of the 28th primer pair are shown in sequence SEQ ID NO: 1-SEQ ID NO: 56.

[0016] The present invention also provides a kit for identifying fast-growing Litopenaeus vannamei populations, the kit containing the aforementioned primer set.

[0017] This invention also provides the application of the MNP marker site, the primer set, or the kit in screening fast-growing Litopenaeus vannamei populations.

[0018] Furthermore, the application specifically includes the following steps:

[0019] S1: Collect Litopenaeus vannamei shrimp to be tested;

[0020] S2: Extract DNA from the Litopenaeus vannamei to be tested, and perform PCR amplification using the primer set described above to obtain the amplification product;

[0021] S3: The amplification product is detected, purified, and sequenced. The obtained sequence is compared with the MNP marker site to obtain the genotyping result of the site.

[0022] Furthermore, in step S3, if the bases at each point of the MNP marker in the sample to be tested are consistent with at least one set of base sequences at the corresponding sites of the fast-growing population in the MNP marker, then the sample is considered to belong to the fast-growing Litopenaeus vannamei population.

[0023] Furthermore, the PCR amplification system in step S2 is as follows: using a 25 μl PCR system, 10-30 ng / μl DNA template, 1 μl forward primer, 10 μM reverse primer, 1 μl reverse primer, 2.5 μl 10×Taq buffer, 0.5 μl dNTPs (10 mM), 1.5 μl MgCl2 (25 mM), 0.25 μl Taq Polymerase (5 U / μl), and 17.25 μl ddH2O.

[0024] Furthermore, the PCR amplification conditions in step S2 are as follows: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 40 s, followed by annealing at a specific temperature of 40 s, extension at 72℃ for 40 s, repeated for a total of 35 cycles; and finally extension at 72℃ for 10 min.

[0025] This invention also provides the application of the MNP marker site, the primer set, or the kit in the identification of kinship and analysis of genetic diversity in Litopenaeus vannamei.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] 1. This invention provides MNP marker loci that can be used to identify Litopenaeus vannamei populations with the trait of rapid growth. The MNP marker loci described in this invention have the characteristics of rich alleles, high resolution, accurate comparison and high efficiency.

[0028] 2. The MNP marker sites described in this invention can accurately compare results under different experimental conditions, further verifying the advantages of accurate comparison and high efficiency of the MNP marker sites provided by this invention, especially the accuracy rate of 100% for identifying the three populations SIS, CP and TOP.

[0029] 3. The MNP marker sites described in this invention can efficiently screen for fast-growing Litopenaeus vannamei populations, enabling high-throughput analysis, avoiding the generation of a large number of non-existent erroneous genotypes, reducing detection costs and analytical complexity, and are of great significance for molecular genetics and genotype analysis of aquatic animals. Attached Figure Description

[0030] Figure 1 Principal component analysis of different populations based on SIS strain-specific MNP marker sites;

[0031] Figure 2 Principal component analysis of different populations based on CP strain-specific MNP marker sites;

[0032] Figure 3 Principal component analysis of MNP marker sites specific to TOP strains for different populations. Detailed Implementation

[0033] The technical solution of the present invention will be further described in detail with reference to the following specific examples.

[0034] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0035] Example 1

[0036] 1. Screening and design of MNP marker sites

[0037] (1) Collect Litopenaeus vannamei from eight commercial strains: Thai CP, Thai TOP, Thai GAS, American SIS Fast, American API, American Primo, American KonaBay, and Mexican Blue Genetics. Collect 30-50 samples from each strain and extract high-quality DNA using a marine animal tissue DNA extraction kit.

[0038] (2) All sample DNA was resequencing at 30 μs using the Illumina NovaSeq platform. The raw reads were quality controlled using the fastp software to obtain clean reads. The clean reads were aligned to the reference genome using the BWA software. The variant sites of all samples were obtained using the sentieon software.

[0039] (3) Use GATK software to further filter variant sites: 1) Parameters are QD < 2.0, FS > 60.0, MQ < 40.0, MQRankSum < -12.5, ReadPosRankSum < -8.0; 2) Screen variant sites with sequencing depth of 3-50; 3) Remove variant sites located in SSR regions; 4) Filter out variant sites with a sample missing rate of more than 20%.

[0040] (4) Screening MNPs: Extract SNPs from the variant sites and construct SNP clusters, i.e., any two adjacent SNPs are less than 50 bp. Based on the SNP clusters, screen for MNP molecular markers in the target strain sample population. The MNP marker site must meet the following criteria: 1) the frequency is 1 in the target strain sample population; 2) it is specific, i.e. the frequency is 0 outside the target strain sample population.

[0041] (5) The specific MNP marker sites of each fast-growing commercial strain are as follows:

[0042] Table 1. MNP marker sites specific to SIS strains

[0043] ;

[0044] ;

[0045] ;

[0046] Table 2 CP strain-specific MNP marker sites

[0047] ;

[0048] ;

[0049] Table 3 TOP strain-specific MNP marker sites

[0050] ;

[0051] .

[0052] (6) Design primers for specific MNP marker sites for each commercial strain with fast growth characteristics. Design amplicon to cover the target site ±100 bp region (total length 200~500 bp). Use Primer 5 software to design PCR primers for MNP marker sites. The PCR primer sequences are shown in SEQ ID NO: 1-SEQ ID NO: 56. Specific primer information is shown in Table 4.

[0053] Table 4. PCR primer information for the three strain-specific MNPs

[0054] ;

[0055] .

[0056] Example 2. Verification Process

[0057] (1) API, CP, EGDA (Ecuador), GAS, Kona Bay, Blue Genetics, Primo, SIS fast-growing, SIS high-resistance and TOP populations (purchased through commercial channels) from different years were collected. The specific year and quantity information are shown in Table 5 below:

[0058] Table 5. Year of Collection and Sample Size for Different Groups

[0059]

[0060] (2) DNA was extracted from the samples using a marine animal genomic DNA extraction kit. The DNA quality was determined by 1.5% agarose gel electrophoresis, and the DNA concentration was determined using a NanoDrop ND spectrophotometer (NanoDrop Technologies). All DNA samples were diluted to 15 ng / μL with Tris-EDTA buffer (pH 8.0) for later use.

[0061] (3) Select all specific MNP primers for the three strains and use a 25 μl PCR system, which contains 1 μl template (10~30 ng / μl), 1 μl forward primer (10 μM), 1 μl reverse primer (10 μM), 2.5 μl 10×Taq buffer, 0.5 μl dNTPs (10 mM), 1.5 μl MgCl2 (25 mM), 0.25 μl Taq Polymerase (5 U / μl), and 17.25 μl ddH2O.

[0062] After adding the sample according to the above system, PCR amplification was performed under the following reaction conditions: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 40 s, followed by annealing at a specific temperature for 40 s, extension at 72℃ for 40 s, for a total of 35 cycles; final extension at 72℃ for 10 min; and storage at 4℃.

[0063] The amplified products were detected by 1.5% agarose gel electrophoresis, and the target fragment was purified and then subjected to Sanger sequencing. The obtained sequences were compared with the MNP reference sequences using Bioedit 7.0.0 software to obtain the genotyping results for these sites.

[0064] (4) The accuracy of identifying the SIS, CP, and TOP populations using the 28 specific MNP marker sites of this invention reached 100%, as shown in the results. Figure 1-3 Principal component analysis was performed on all collected samples based on the specific MNP marker sites of the three strains, which clearly distinguished whether the different strains belonged to the fast-growing Litopenaeus vannamei population.

[0065] Example 3. Application Method

[0066] DNA was extracted from samples of unknown origin using a marine animal genomic DNA extraction kit. DNA quality was assessed by 1.5% agarose gel electrophoresis, and DNA concentration was determined using a NanoDrop ND spectrophotometer (NanoDrop Technologies). All DNA samples were diluted to 15 ng / μL with Tris-EDTA buffer (pH 8.0) for later use.

[0067] Using the primer set corresponding to the specific MNP marker site, a 25 μl PCR system was prepared, containing 1 μl DNA template (10~30 ng / μl), 1 μl forward primer (10 μM), 1 μl reverse primer (10 μM), 2.5 μl 10×Taq buffer, 0.5 μl dNTPs (10 mM), 1.5 μl MgCl2 (25 mM), 0.25 μl Taq Polymerase (5 U / μl), and 17.25 μl ddH2O.

[0068] After adding the sample DNA according to the above system, PCR amplification was performed under the following reaction conditions: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 40 s, followed by annealing at the specific temperature for 40 s, extension at 72℃ for 40 s, for a total of 35 cycles; final extension at 72℃ for 10 min; and storage at 4℃.

[0069] The amplified products were detected by 1.5% agarose gel electrophoresis, and the target fragment was purified and then subjected to Sanger sequencing. The obtained sequences were compared with the MNP reference sequences using Bioedit 7.0.0 software to obtain the genotyping results for these sites.

[0070] The genotyping results of all MNP markers in each sample to be tested are compared with the MNP marker sites. If at least one set of the base sequence of some MNP markers in the sample to be tested matches the corresponding site of the fast-growing population in the MNP markers, then the sample is considered to belong to the fast-growing Litopenaeus vannamei population.

[0071] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A primer set for identifying fast-growing Litopenaeus vannamei populations, characterized in that, The primer set is used to detect MNP marker sites associated with rapid growth. The sequences of the primer set are shown in SEQ ID No. 1-SEQ ID No. 56, and the MNP marker sites are shown in Tables 1, 2 and 3. Table 1. MNP marker sites specific to SIS strains ， ， Table 2 CP strain-specific MNP marker sites ， ， ， Table 3 TOP strain-specific MNP marker sites ， 。 2. A kit for identifying fast-growing Litopenaeus vannamei populations, characterized in that, The kit contains the primer set as described in claim 1.

3. The application of the primer set of claim 1 or the kit of claim 2 in screening fast-growing Litopenaeus vannamei populations.

4. The application according to claim 3, characterized in that, The application specifically includes the following steps: S1: Collect Litopenaeus vannamei shrimp to be tested; S2: Extract DNA from the Litopenaeus vannamei to be tested, and perform PCR amplification using the primer set described in claim 1 to obtain the amplification product; S3: The amplification product is detected, purified, and sequenced. The obtained sequence is compared with the MNP marker site to obtain the genotyping result of the site.

5. The application according to claim 4, characterized in that, In step S3, if the base sequence at each point of the MNP marker in the sample to be tested is consistent with the base sequence at the corresponding site of the fast-growing population in the MNP marker of claim 1, then the sample is considered to belong to the fast-growing Litopenaeus vannamei population.

6. The application according to claim 4, characterized in that, The PCR amplification system in step S2 is as follows: a 25 μl PCR system, 10-30 ng / μl DNA template, 1 μl forward primer, 10 μM reverse primer, 1 μl reverse primer, 2.5 μl 10×Taq buffer, 0.5 μl dNTPs, 1.5 μl MgCl2, 1.5 μl 25 mM MgCl2, 0.25 μl Taq Polymerase, 0.25 μl 5 U / μl ddH2O, and 17.25 μl ddH2O.

7. The application according to claim 4, characterized in that, The PCR amplification conditions in step S2 are as follows: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 40 s, followed by annealing at a specific temperature of 40 s, extension at 72℃ for 40 s, repeated for a total of 35 cycles; and final extension at 72℃ for 10 min.

8. The application of the primer set of claim 1 or the kit of claim 2 in the identification of kinship and analysis of genetic diversity in Litopenaeus vannamei.

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