MNP marker site and primer group for identifying rapid growth type litopenaeus vannamei population and application of MNP marker site and primer group

Abstract

The invention provides an MNP marker site for identifying a rapid growth type litopenaeus vannamei population, a primer group and application of the MNP marker site and the primer group, and belongs to the technical field of molecular genetics of aquatic animals. The MNP marker site, the corresponding primer group and the kit thereof can screen a litopenaeus vannamei population with the character of high growth speed, and prove that the MNP marker site can realize high-throughput analysis, avoid the generation of a large number of nonexistent error genotypes, reduce the detection cost and analysis complexity, and improve the detection efficiency. And the accuracy rate of identifying the rapid growth type litopenaeus vannamei population is up to 100%. The MNP marker site and application provided by the invention have reference significance in the related fields of molecular inheritance and genotype analysis of aquatic animals.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic animal molecular genetics, and particularly relates to an MNP marker site, a primer set and an application thereof for identifying a fast-growing Litopenaeus vannamei population. Background Art

[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, trait expression, and adaptability. For example, strains such as the US SIS (Shrimp Improvement System), Thailand's CP (Chia Tai), and Thailand's TOP (Top Feng) are characterized by fast growth, while strains such as the US API (American Shrimp Company), US Primo (Primo), and Mexico's Blue Genetics are highly disease-resistant. The US Kona Bay strain combines both fast growth and high disease resistance. The lack of accurate identification methods can easily lead to problems such as germplasm contamination, cultivar infringement, and inbreeding depression, directly impacting aquaculture profitability and the sustainable development of the industry. Genetic identification of commercial stocks abroad using specific molecular markers can ensure the purity of introduced stocks and avoid production risks associated with germplasm contamination, thereby safeguarding the rights of breeders and the security of the seed 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, with limited single-time detection sites, making it difficult to achieve high-throughput analysis. There are many manual steps and it is easy to introduce human errors. When polymerase amplifies simple repeat sequences in SSR markers, there is a "slippage" phenomenon, resulting in a large number of non-existent erroneous genotypes. A single SNP only provides bi-allelic information, and a large number of sites (usually hundreds to thousands) must be combined to achieve high-precision identification, which increases the detection cost and analysis complexity. Mainstream SNP typing technologies (such as chips and sequencing) require expensive instruments and bioinformatics analysis capabilities, which are not friendly to small-scale laboratories. In this regard, multi-nucleotide polymorphism (MNP) markers are a more ideal identification method with the following advantages: (1) Rich allele types and high resolution. MNP markers have up to 2 n The MNP markers are designed to generate a set of alleles (where n is the number of SNP markers in the MNP); (2) MNP marker typing results are standardized, allowing accurate comparison between results obtained under different experimental conditions; and (3) they are highly efficient. A single PCR reaction can amplify several to dozens of SNP loci. The development of population-specific MNP markers can facilitate the identification of commercial populations of Litopenaeus vannamei with the important fast-growing trait. Summary of the Invention

[0004] The present invention aims to provide an MNP marker site, a primer set, and applications thereof for identifying fast-growing Litopenaeus vannamei populations. The MNP marker site screened by the present invention can screen for fast-growing Litopenaeus vannamei populations. The MNP marker site can achieve high-throughput analysis, avoid generating a large number of erroneous genotypes that do not exist, reduce detection costs and analysis complexity, and has reference significance for the fields related to molecular genetics and genotyping analysis of aquatic animals.

[0005] In order to achieve the above invention objectives, the present invention is implemented through the following technical solutions: The present invention provides an MNP marker site for identifying a fast-growing population of Penaeus vannamei. The location of the MNP marker site is as follows: ; ; ; ; ; ; .

[0006] The present invention also provides a primer set for identifying a fast-growing population of Penaeus vannamei. The primer set is used to detect the MNP marker site. The sequences of the primer set are shown in SEQ ID No. 1 to SEQ ID No. 56.

[0007] Furthermore, the primer set includes: the 1st primer pair to the 28th primer pair, each primer pair includes a forward primer and a reverse primer, and the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the forward primer of the 28th primer pair and the reverse primer of the 28th primer pair are shown in the sequences SEQ ID NO: 1 to SEQ ID NO: 56, respectively.

[0008] The present invention also provides a kit for identifying a fast-growing Litopenaeus vannamei population, and the kit contains the primer set.

[0009] The present invention also provides the use of the MNP marker site, the primer set or the kit in screening a fast-growing Litopenaeus vannamei population.

[0010] Furthermore, the application specifically includes the following steps: S1: Collect the shrimp to be tested; S2: extracting DNA from the tested Litopenaeus vannamei, and performing PCR amplification using the primer set to obtain an amplified product; S3: Detecting, purifying and sequencing the amplified product, comparing the obtained sequence with the MNP marker site, and obtaining the typing result of the site.

[0011] Furthermore, in step S3, if the bases at each site of the MNP marker in the sample to be tested are consistent with the base sequences of the corresponding sites of the fast-growing population in the MNP marker after at least one group is aligned, the sample is considered to belong to the fast-growing population of Penaeus vannamei.

[0012] Furthermore, the PCR amplification system in step S2 is as follows: using a 25 μl PCR system, a DNA template of 10-30 ng / μl, 1 μl, a forward primer of 10 μM, 1 μl, a reverse primer of 10 μM, 1 μl, 2.5 μl of 10×Taq buffer, 0.5 μl of 10 mM dNTPs, 1.5 μl of 25 mM MgCl2, 0.25 μl of Taq Polymerase 5 U / μl, and 17.25 μl of ddH2O.

[0013] Furthermore, the PCR amplification conditions in step S2 are: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 40 s, annealing at a specific temperature for 40 s, extension at 72°C for 40 s, repeated for a total of 35 cycles; and final extension at 72°C for 10 min.

[0014] The present invention also provides the use of the MNP marker site, the primer set or the kit in the identification of kinship and analysis of genetic diversity of Litopenaeus vannamei.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides an MNP marker locus that can be used to identify populations of Penaeus vannamei with fast-growing traits. The MNP marker locus of the present invention has the characteristics of rich allele types, high resolution, accurate comparison, and high efficiency.

[0016] 2. The MNP-labeled sites described in the present invention enable accurate comparison of results obtained under different experimental conditions, further validating the advantages of the MNP-labeled sites provided by the present invention in terms of precise comparison and high efficiency, especially for the identification of the three populations of SIS, CP, and TOP, with an accuracy rate of up to 100%.

[0017] 3. The MNP marker loci described in the present invention can efficiently screen for fast-growing populations of Litopenaeus vannamei, enabling high-throughput analysis while avoiding the generation of a large number of non-existent erroneous genotypes, reducing detection costs and analysis complexity, and are of great significance for molecular genetic and genotyping analysis of aquatic animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Principal component analysis of SIS strain-specific MNP marker loci for different groups; Figure 2 Principal component analysis based on CP strain-specific MNP marker loci for different groups; Figure 3 Principal component analysis of TOP strain-specific MNP marker loci for different groups. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0020] In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0021] Example 1 1. Screening and design of MNP labeling sites (1) Eight commercial strains of Penaeus vannamei, including CP from Thailand, TOP from Thailand, GAS from Thailand, SIS from the United States, API from the United States, Primo from the United States, KonaBay from the United States, and Blue genetics from Mexico, were collected. 30 to 50 samples were collected from each strain, and high-quality DNA was extracted using a marine animal tissue DNA extraction kit.

[0022] (2) All sample DNA was resequenced at 30× using the Illumina NovaSeq platform. Raw reads were quality controlled using fastp software to obtain clean reads. Clean reads were aligned to the reference genome using BWA software. Sentieon software was used to obtain variant sites for all samples.

[0023] (3) GATK software was used to further filter variant sites: 1) the parameters were QD < 2.0, FS > 60.0, MQ < 40.0, MQRankSum < -12.5, and ReadPosRankSum < -8.0; 2) variant sites with a sequencing depth of 3-50 were screened; 3) variant sites located in the SSR region were removed; 4) variant sites with a sample missing rate higher than 20% were filtered out.

[0024] (4) Screening for MNPs: Extract SNPs from the variant sites and construct a SNP cluster, i.e., any two adjacent SNPs are less than 50 bp apart. Based on the SNP cluster, screen for MNP molecular markers in the target strain sample group. The MNP marker site must meet the following criteria: 1) The frequency within the target strain sample group is 1; 2) It is specific, i.e., the frequency outside the target strain sample group is 0.

[0025] (5) The specific MNP marker site information of each fast-growing commercial strain is as follows: Table 1. SIS strain-specific MNP marker sites ; ; ; Table 2 CP strain-specific MNP marker sites ; ; Table 3 TOP strain-specific MNP marker sites ; .

[0026] (6) Primers were designed for the specific MNP marker sites of each fast-growing commercial variety. The amplicon was designed to cover the ±100 bp region of the target site (total length 200-500 bp). PCR primers for the MNP marker sites were designed using Primer 5 software. The PCR primer sequences are shown in SEQ ID NO: 1-SEQ ID NO: 56. Specific primer information is shown in Table 4.

[0027] Table 4 PCR primer information for three strain-specific MNPs ; .

[0028] Example 2. Verification process (1) We collected populations of API, CP, EGDA (Ecuador), GAS, Kona Bay, Blue genetics, Primo, SIS fast-growing, SIS high-resistance, and TOP from different years (purchased from commercial sources). The specific years and quantities are shown in Table 5 below: Table 5. Collection years and sample numbers of different groups

[0029] (2) DNA was extracted from the samples using a marine animal genomic DNA extraction kit. DNA quality was determined by 1.5% agarose gel electrophoresis, and DNA concentration was measured using a NanoDrop ND spectrophotometer (NanoDrop Technologies). All DNA samples were diluted to 15 ng / μL using Tris-EDTA buffer (pH 8.0) for later use.

[0030] (3) All specific MNP primers of the three strains were selected and a 25 μl PCR system was used, including 1 μl of template (10-30 ng / μl), 1 μl of forward primer (10 μM), 1 μl of reverse primer (10 μM), 2.5 μl of 10× Taq buffer, 0.5 μl of dNTPs (10 mM), 1.5 μl of MgCl2 (25 mM), 0.25 μl of Taq Polymerase (5 U / μl), and 17.25 μl of ddH2O.

[0031] After adding the sample according to the above system, PCR amplification was performed according to the following reaction conditions: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 40 s, annealing at a specific temperature for 40 s, extension at 72°C for 40 s, repeated 35 cycles in total; final extension at 72°C for 10 min; and storage at 4°C.

[0032] Amplified products were detected by 1.5% agarose gel electrophoresis, and the target fragments were purified and then subjected to Sanger sequencing. The obtained sequences were aligned with the MNP reference sequence using Bioedit 7.0.0 software to obtain the typing results for these loci.

[0033] (4) The accuracy of identifying the three groups of SIS, CP and TOP using the 28 specific MNP marker sites of the present invention reached 100%, as shown in the following results: Figure 1-3 Principal component analysis of all collected samples based on the specific MNP marker sites of the three strains can clearly distinguish whether different strains belong to the fast-growing vannamei population.

[0034] Example 3. Application method DNA from samples of unknown origin was extracted 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 in Tris-EDTA buffer (pH 8.0) before use.

[0035] The primer set corresponding to the specific MNP labeling site was selected, and a 25 μl PCR system was used, including 1 μl of DNA template (10-30 ng / μl), 1 μl of forward primer (10 μM), 1 μl of reverse primer (10 μM), 2.5 μl of 10× Taq buffer, 0.5 μl of dNTPs (10 mM), 1.5 μl of MgCl2 (25 mM), 0.25 μl of Taq Polymerase (5 U / μl), and 17.25 μl of ddH2O.

[0036] After adding the sample DNA according to the above system, PCR amplification was performed according to the following reaction conditions: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 40 s, annealing at a specific temperature for 40 s, extension at 72°C for 40 s, repeated 35 cycles in total; final extension at 72°C for 10 min; and storage at 4°C.

[0037] Amplified products were detected by 1.5% agarose gel electrophoresis, and the target fragments were purified and then subjected to Sanger sequencing. The obtained sequences were aligned with the MNP reference sequence using Bioedit 7.0.0 software to obtain the typing results for these loci.

[0038] The typing results of all MNP markers of each sample to be tested are compared with the MNP marker sites. If at least one group of base sequences of some MNP markers of the sample to be tested is consistent with the base sequences of the sites corresponding to the fast-growing population in the MNP marker, the sample is considered to belong to the fast-growing population of Penaeus vannamei.

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A MNP marker site for identifying a population of fast-growing Litopenaeus vannamei, characterized in that: The MNP labeling sites are shown in Table 1, Table 2 and Table 3: Table 1. SIS strain-specific MNP marker sites ； ； Table 2 CP strain-specific MNP marker sites ； ； ； Table 3 TOP strain-specific MNP marker sites ； 。 2. A primer set for identifying a population of fast-growing Litopenaeus vannamei, characterized in that: The primer set is used to detect the MNP labeling site according to claim 1, and the sequence of the primer set is shown as SEQ ID No. 1 to SEQ ID No.

56.

3. A kit for identifying fast-growing Litopenaeus vannamei populations, characterized in that: The kit contains the primer set according to claim 2.

4. Use of the MNP marker site according to claim 1, the primer set according to claim 2, or the kit according to claim 3 in screening a population of fast-growing Litopenaeus vannamei.

5. The use according to claim 4, characterized in that The application specifically includes the following steps: S1: Collect the shrimp to be tested; S2: extracting DNA from the tested Litopenaeus vannamei, and performing PCR amplification using the primer set of claim 2 to obtain an amplified product; S3: Detecting, purifying and sequencing the amplified product, comparing the obtained sequence with the MNP marker site, and obtaining the typing result of the site.

6. The use according to claim 5, characterized in that In step S3, if the bases at each site of the MNP marker in the sample to be tested are consistent with the base sequences of the corresponding sites of the fast-growing population in the MNP marker according to claim 1 after alignment, the sample is considered to belong to the fast-growing population of Penaeus vannamei.

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

8. The use according to claim 5, characterized in that The PCR amplification conditions in step S2 are as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 40 s, annealing at a specific temperature for 40 s, extension at 72°C for 40 s, repeated for 35 cycles; and final extension at 72°C for 10 min.

9. Use of the MNP marker site according to claim 1, the primer set according to claim 2, or the kit according to claim 3 in the identification of kinship and analysis of genetic diversity of Litopenaeus vannamei.

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