Litopenaeus vannamei TAUD gene, SNP marker, detection primer, kit and application of litopenaeus vannamei TAUD gene

Abstract

The invention discloses a litopenaeus vannamei TAUD gene, an SNP marker, a detection primer, a kit and application of the litopenaeus vannamei TAUD gene. The sequence of the SNP marker is as shown in SEQ ID NO.1, the SNP site is located at the 504th site from the 5'end of the SEQ ID NO.1, and the polymorphic form is T / C type; the SNP site can be used for simultaneously screening body length and body weight to breed litopenaeus vannamei, and a primer designed according to the sequence of SEQ ID NO.1 or a kit prepared from the primer can be used for detecting the SNP site. The TT genotype of the SNP marker Chr2414603628 provided by the invention is used for selecting parents, so that the body length growth speed of a breeding population can be increased, an innovative tool is provided for molecular breeding of litopenaeus vannamei, the breeding period is shortened, and industrial development and efficiency improvement are assisted.

Description

Technical Field

[0001] This invention relates to the fields of aquatic biotechnology and molecular breeding, specifically to a Litopenaeus vannamei TAUD gene, SNP marker, detection primers, kit, and their applications. Background Technology

[0002] Litopenaeus vannamei ( Penaeus vannamei Litopenaeus vannamei is one of the most important economic species in global aquaculture, widely promoted for cultivation due to its rapid growth, strong environmental adaptability, and high economic value. However, aquaculture practices show that there are significant individual differences in the growth performance of Litopenaeus vannamei. Shrimp growth traits are jointly regulated by genetic and environmental factors, with body weight and growth rate being key indicators for measuring their economic value; low levels of either directly impact aquaculture efficiency and industry profits.

[0003] Currently, the breeding of Litopenaeus vannamei mainly relies on traditional selection methods. Although these methods can improve growth traits to some extent, they still have the following limitations: (1) Long breeding cycle and low efficiency: Traditional methods rely on phenotypic selection, but since growth traits are quantitative traits and are easily affected by the environment, the selection process is slow and it is difficult to adapt to the rapidly growing market demand; (2) Lack of precise molecular markers: Although technologies such as genome selection and molecular marker-assisted selection have been gradually applied to shrimp breeding, efficient and specific molecular markers for growth traits are still scarce. Existing markers often have a wide coverage and cannot accurately point to genetic loci that are highly related to growth, which limits the efficiency of genetic progress; (3) Difficulty in achieving precise genetic improvement: The genetic structure of growth traits in Litopenaeus vannamei is complex. Traditional phenotypic selection is easily affected by environmental fluctuations and it is difficult to stably screen and transmit superior growth genes.

[0004] Single nucleotide polymorphisms (SNPs), as third-generation molecular markers, possess advantages such as high density, strong stability, and co-dominance, and have become the most widely used molecular marker technology in the breeding of economically important crustaceans such as shrimp and crab. However, the discovery of SNPs in genes related to growth traits in Litopenaeus vannamei is relatively limited, and most SNP markers regulate only one growth trait, making it impossible to screen for two or more growth traits simultaneously. Therefore, it is still necessary to further explore and systematically validate SNPs in genes related to growth traits using high-throughput omics methods to promote the construction of a precise and efficient molecular breeding system.

[0005] TAUD is a type of Fe 2+ A 2-OG-dependent dioxygenase, TAUD primarily catalyzes the oxidative cleavage of the CS bond in taurine to generate sulfite, which is used in sulfur metabolism and cellular redox reactions. The TAUD protein contains a conserved CAS-like / TauD domain and belongs to the Fe... 2+The α-ketoglutarate-dependent dioxygenase superfamily possesses a typical catalytic core and has identified multiple conserved iron-binding sites and active sites. In Litopenaeus vannamei, the relationship between the TAUD gene and its upstream regulatory regions and shrimp growth traits has not yet been investigated.

[0006] Therefore, developing a single SNP marker that simultaneously screens for body length and weight growth traits in Litopenaeus vannamei targeting the TAUD gene and its upstream regulatory regions is of great significance for the breeding of new Litopenaeus vannamei varieties. It provides an innovative tool for molecular breeding of Litopenaeus vannamei, adding screening criteria to the traditional single SNP marker screening of a single growth trait, making SNP marker screening more accurate, further shortening the breeding cycle, and contributing to industrial development and efficiency improvement. Summary of the Invention

[0007] The purpose of this invention is to develop a TAUD gene, SNP marker, detection primers, kit, and their applications for Litopenaeus vannamei. This allows a single SNP marker developed from the TAUD gene and its upstream regulatory regions to simultaneously screen for body length and weight growth traits in Litopenaeus vannamei. This adds a screening criterion to the traditional method of using a single SNP marker to screen for a single growth trait, making SNP marker screening more precise, accelerating the breeding and genetic improvement of fast-growing Litopenaeus vannamei varieties, and improving breeding efficiency and economic benefits.

[0008] To solve this technical problem: This patent provides an SNP marker, the sequence of which is shown in SEQ ID NO.1. The SNP site is located at position 504 from the 5' end of the sequence shown in SEQ ID NO.1, and the polymorphism of the SNP marker is T / C type.

[0009] Another aspect of this patent provides the application of SNP markers in the breeding of body length and weight of Litopenaeus vannamei. The sequence of the SNP marker is shown in SEQ ID NO.1. The SNP site is located at position 504 from the 5' end of the sequence shown in SEQ ID NO.1. The polymorphism of the SNP marker is T / C type. The SNP marker can simultaneously screen for body length and weight of Litopenaeus vannamei.

[0010] Furthermore, the SNP marker is located in the upstream 5' regulatory region of the TAUD gene, the sequence of which is shown in SEQ ID NO.2.

[0011] Another aspect of this patent provides a primer pair for detecting SNP markers, the base sequence of which is: Forward primer F: 5'-AATCCGCCACTGAGCCAATTA-3'; Reverse primer R: 5'-GAAGGCCGAGTAACCGACTT-3'.

[0012] Another aspect of this patent provides the application of primer pairs for detecting SNP markers in the breeding of body length and weight of Litopenaeus vannamei. The base sequence of the primer pair is: Forward primer F: 5'-AATCCGCCACTGAGCCAATTA-3'; Reverse primer R: 5'-GAAGGCCGAGTAACCGACTT-3'; The sequence of the SNP marker is shown in SEQ ID NO.1. The SNP site is located at position 504 from the 5' end of the sequence shown in SEQ ID NO.1. The polymorphism of the SNP marker is T / C type.

[0013] Another aspect of this patent provides an SNP labeling kit, including primer pairs, the base sequences of which are: Forward primer F: 5'-AATCCGCCACTGAGCCAATTA-3'; Reverse primer R: 5'-GAAGGCCGAGTAACCGACTT-3'.

[0014] Another aspect of this patent provides an application of an SNP marker kit in the breeding of body length and weight in Litopenaeus vannamei. The kit includes primer pairs, the base sequences of which are: Forward primer F: 5'-AATCCGCCACTGAGCCAATTA-3'; Reverse primer R: 5'-GAAGGCCGAGTAACCGACTT-3'; The sequence of the SNP marker is shown in SEQ ID NO.1. The SNP site is located at position 504 from the 5' end of the sequence shown in SEQ ID NO.1. The polymorphism of the SNP marker is T / C type.

[0015] This patent also provides a method for breeding Litopenaeus vannamei, including the following steps: Step S1: Detect the SNP molecular markers of Litopenaeus vannamei to obtain the genotype of the SNP site; the sequence of the SNP marker is shown in SEQ ID NO.1; the SNP site is located at position 504 from the 5' end of the sequence shown in SEQ ID NO.1; the polymorphism of the SNP molecular marker is T / C type; Step S2: Simultaneously screen Litopenaeus vannamei for body length and weight based on the genotype of the SNP locus; the genotype of the SNP locus is TT, TC, or CC. Step S3: Use Litopenaeus vannamei male and female parents with SNP locus genotype TT for crossbreeding.

[0016] Furthermore, step S1 includes: Step S1-1: Extract genomic DNA from Litopenaeus vannamei; Step S1-2: Using the genomic DNA of Litopenaeus vannamei as a template, PCR amplification was performed using the following primer pairs to obtain the PCR amplification product; Forward primer F: 5'-AATCCGCCACTGAGCCAATTA-3'; Reverse primer R: 5'-GAAGGCCGAGTAACCGACTT-3'; Steps S1-3: Sequencing the PCR amplification products to obtain the genotype of the SNP site. The genotype of the SNP site is the T / C type at position 504 from the 5' end of the sequence shown in SEQ ID NO.1.

[0017] Furthermore, in steps S1-2, The PCR amplification system consisted of a 25 μL amplification system, which included: 1 μL of Litopenaeus vannamei genomic DNA at a concentration of 100 ng / μL; 1 μL of forward primer F at a concentration of 10 μM; 1 μL of reverse primer R at a concentration of 10 μM; 12.5 μL of 2×Taq Plus Master Mix II; and 9.5 μL of enzyme-free sterile water.

[0018] Furthermore, the PCR amplification program includes the following steps: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at 56℃ for 20 s, extension at 72℃ for 40 s, for a total of 32 cycles from denaturation to extension, followed by a full extension at 72℃ for 10 min.

[0019] Furthermore, in step S2, the genotypes of SNP sites are selected based on the following criteria: body length and weight of Litopenaeus vannamei individuals with the TT genotype at the SNP site > body length and weight of Litopenaeus vannamei individuals with the TC genotype at the SNP site > body length and weight of Litopenaeus vannamei individuals with the CC genotype at the SNP site.

[0020] Furthermore, step S3 specifically involves retaining Litopenaeus vannamei with the TT genotype at the SNP locus and breeding them by pairing male and female Litopenaeus vannamei with the TT genotype.

[0021] Another aspect of this patent provides a method for reducing the expression of the TAUD gene in Litopenaeus vannamei. The method specifically involves injecting siTAUD-dsRNA into the middle of the second to third abdominal segments of Litopenaeus vannamei at a dose of 2 μg / g / shrimp for more than 20 days, which can significantly reduce the expression level of the TAUD gene in Litopenaeus vannamei.

[0022] Furthermore, siTAUD-dsRNA is obtained by designing siRNA sequence pairs targeting the TAUD gene, and then synthesizing dsRNA in vitro after dissolving in enzyme-free sterile water.

[0023] Furthermore, the siRNA sequence pairs are: siTAUD-F: 5'-TATGCATGGAAAGGAAGGTGTCATAAGGAT-3'; siTAUD-R: 5'-CTGTTTCAGAGAGCAGATTGACATCAAC-3'.

[0024] Therefore, it can be concluded that this invention locates a body length-related SNP molecular marker site in the 5' regulatory region upstream of the TAUD gene in Litopenaeus vannamei through genome-wide association analysis. The sequence of the Chr24_14603628 molecular marker is shown in SEQ ID NO.1, and the SNP site is located at position 504 from the 5' end of the sequence shown in SEQ ID NO.1, exhibiting a T / C polymorphism. Primers SEQ ID NO.3 and SEQ ID NO.4, designed based on the sequence in SEQ ID NO.1, can detect the Chr24_14603628 molecular marker, and these primers can also be prepared into a kit. Simultaneously, the siRNA sequence targeting the TAUDE gene, when injected in vitro into Litopenaeus vannamei, significantly inhibits the body length growth rate of the shrimp. Subsequent overexpression of the TAUD gene in Litopenaeus vannamei can achieve rapid body length growth. Therefore, the SNP molecular marker Chr24_14603628 provided by this invention can be used to assist in the selection of fast-growing parents. By using the TT or TC genotype in this SNP molecular marker to select parents, the growth rate of the breeding population can be improved, providing an innovative tool for molecular breeding of Litopenaeus vannamei, shortening the breeding cycle, and contributing to industrial development and efficiency improvement. Attached Figure Description

[0025] The above description of the present invention and the following detailed embodiments will be better understood when read in conjunction with the accompanying drawings. It should be noted that the drawings are merely examples of the claimed technical solutions.

[0026] Figure 1The Manhattan plot for genome-wide association analysis of body length in Litopenaeus vannamei in this example (where the x-axis represents chromosomes and the y-axis represents significance, using -log) 10 (P) indicates); Figure 2 This is a fine-mapping analysis diagram of Litopenaeus vannamei after meta-GWAS in the example (where A is the meta-GWAS result, the horizontal axis is chromosome localization, and the vertical axis is significance, calculated using -log). 10 (P) represents the fine mapping of locus Chr24_14603628, with the horizontal axis representing chromosome location and the vertical axis representing posterior inclusion probability, represented using PIP). Figure 3 The figure shows the distribution of body length and weight of different genotypes of Litopenaeus vannamei in the example (where the horizontal axis represents genotype, with CC type represented in green, TC type in red, and CC type in blue; the vertical axis of the left figure represents body length (mm), and the vertical axis of the right figure represents weight (g); " "P-value" indicates a significant difference in individual traits between the two genotypes of Litopenaeus vannamei; "NS" indicates no significant difference in individual traits between the two genotypes of Litopenaeus vannamei. Figure 4 The image shown is an agarose gel electrophoresis image of the genomic DNA of Litopenaeus vannamei in the example (where M is the marker; 1 is the blank control; 2-16 are the target genes, all of which are over 5000 bp in length and have good integrity). Figure 5 The image shows an agarose gel electrophoresis image of the PCR amplification products of Litopenaeus vannamei in the example (where M is the marker; 1 is the blank control; and 2-16 are the PCR products of the target sequence, each 878 bp in length). Figure 6 This is a flowchart illustrating the sequence sequencing process in the example. Figure 7 The sequencing results of the Litopenaeus vannamei PCR amplification products in the example are shown in the peak diagram (where the genotype at position 451 is the target genotype). Figure 8 The image shows the body length of Litopenaeus vannamei after RNA interference in the examples (where the horizontal axis represents body length in cm; the vertical axis represents different RNA interference levels, with blue representing the negative control group (siNC) and orange representing the experimental group (siTAUD)). "This indicates that there are significant differences in individual traits between the two groups of Litopenaeus vannamei." Figure 9 The image shown is an agarose gel electrophoresis image of total RNA from the intestine of Litopenaeus vannamei after RNA interference in the example (where M is the marker, and 1-24 are all total RNA after RNA interference). Figure 10 This is a graph showing the relative expression level of the TAUD gene in the intestine of Litopenaeus vannamei after RNA interference in the examples (where the horizontal axis represents the negative control group (siNC) and the experimental group (siTAUD); the vertical axis represents the relative expression level). "This indicates that the P-value shows a significant difference in body length between the two groups of Litopenaeus vannamei individuals." Detailed Implementation

[0027] The detailed features and advantages of this application are described below in the specific embodiments. The content of this description is sufficient to enable any person skilled in the art to understand the technical content of this application and implement it accordingly. Based on the specification, claims and drawings disclosed in this specification, a person skilled in the art can easily understand the related objectives and advantages of this application.

[0028] In this specification and claims, several terms will be used, and unless otherwise indicated, these terms will be defined to have the following meanings: All other terms used herein for special definition are intended to have the general meaning understood by one of ordinary skill in the art, and in particular, meaning that one of ordinary skill in the art, upon reading the claims, specification and drawings of this patent, can directly and without doubt determine how the technical solution of this patent can be implemented.

[0029] Even if there are incomplete descriptions, omissions, or ambiguities in the grammar, words, punctuation, graphics, symbols, etc. of the claims, specification, and drawings of this patent, a person skilled in the art can still arrive at the only correct understanding by reading the claims, specification, and drawings as a whole without extensive reasoning or experimentation, and effectively exclude various incorrect interpretations that are not aimed at achieving the purpose of this patent.

[0030] The "range" disclosed herein is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0031] Unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0032] Unless otherwise specified, all the technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0033] Unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0034] Unless otherwise specified, the terms "comprising" and "including" as used herein can be open-ended or closed-ended. For example, "comprising" and "including" may mean that other components not listed may also be included, or that only the listed components may be included.

[0035] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.

[0036] In this description, unless otherwise stated, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0037] Unless otherwise specified, percentages (%) in this document refer to percentages by mass relative to the composition.

[0038] Unless otherwise stated herein, the sum of the contents of the components in the composition is 100%.

[0039] In this document, unless otherwise stated, “combination of” means a multi-component mixture of the elements, such as two, three, four, and up to the maximum possible multi-component mixture.

[0040] Unless otherwise specified, the term "a" as used in this specification means "at least one".

[0041] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0043] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0044] (1) Source of sample materials In this embodiment, Litopenaeus vannamei was purchased from Hainan Renhai Aquatic Products Technology Co., Ltd. (Wenchang City, Hainan Province).

[0045] In this embodiment, primer synthesis was performed by Beijing Qingke Biotechnology Co., Ltd., and sequencing was performed by Beijing Novogene Technology Co., Ltd.

[0046] (2) Sources of reagents and consumables Table 1. Sources and Product Codes of Reagents and Consumables Required for the Experiment ;

[0047] (3) Source of instruments and equipment Table 2. Sources and Models of Instruments and Equipment Required for the Experiment ;

[0048] Example This patent provides a method for detecting the TAUD gene, SNP markers, primers, and reagent kits in Litopenaeus vannamei, and their applications, including the following steps: S1. Assembly of the reference genome, the specific steps are as follows: The genome of Litopenaeus vannamei was assembled using a third-generation sequencing system based on the Oxford Nanopore Technologies (ONT) platform. This assembly has not yet been registered with NCBI. The specific assembly steps are as follows: 1. In terms of experimental procedures, muscle tissue from Litopenaeus vannamei was used as the sample. After collection, the samples were rapidly frozen in liquid nitrogen and then transferred to a -80°C environment for long-term storage to ensure the integrity of the tissue's RNA and DNA molecules and prevent degradation that could affect subsequent extraction of ultra-long DNA fragments. Based on this, total genomic DNA was extracted using a traditional, gentle nuclear method (commercially available kit, following the kit instructions). High-quality DNA samples with a main band greater than 100 kb were rigorously screened using pulsed-field electrophoresis quality control. Only DNA samples meeting this ultra-long fragment standard were allowed to proceed to the library construction stage, a crucial prerequisite for achieving ultra-long sequencing reads.

[0049] 2. Library construction utilized the ONT SQK-LSK114 ligation sequencing kit (version 14, commercially available). This kit is deeply optimized for the R10.4.1 sequencing chip, effectively improving single-base discrimination accuracy while maintaining ultra-long read lengths, especially for common homopolymer regions in the genome. Sequencing was performed on an R10.4.1 flow cell, which, with its advanced protein channel design and electronics, provides hardware support for high-throughput, high-quality data output. After the raw sequencing current signal was processed, it was converted using the dna_r10.4.1_e8.2_400bps_sup@v5.0.0 high-precision base recognition model in Doradov 0.9.0 software. This model employs the latest deep learning algorithm, achieving super-accuracy base discrimination, significantly reducing sequencing noise, and providing near-perfect accuracy input data for genome assembly and subsequent structural variation analysis.

[0050] 3. The raw sequencing signals were converted using Dorado v0.9.0 to obtain FASTQ format read sequences. NanoPlot was used for read distribution statistics, filtering out short reads <10 kb in length, and extracting high-confidence reads with a base quality >Q20 for subsequent analysis. After quality control, approximately 181 Gb of raw data was obtained, of which approximately 117 Gb was high-quality data above Q20. NextDenovo v2.5.0 was used to self-correct long read sequences above Q20. Through cross-read alignment and consistency calculation, random errors and chimeric sequences in the raw reads were corrected, resulting in approximately 104 Gb of high-confidence corrected reads. Considering the highly repetitive nature of the Litopenaeus vannamei genome, ultra-long reads >20 kb in length were further screened before formal assembly, resulting in approximately 90 Gb of high-quality dataset. The NextDenovo assembly module was used for de novo assembly, fully leveraging the advantage of ultra-long reads traversing complex repetitive regions to ensure the continuity of genome assembly. NextPolish v1.4.0 was used to polish the preliminary assembly results in multiple rounds: (1) the original ultra-long reads were aligned back to the assembly sequence to correct insertion and deletion errors; (2) more than two rounds of base-level error correction were performed in combination with second-generation short read data to finally obtain a high-fidelity genome draft.

[0051] 4. A multi-strategy comprehensive evaluation was adopted: (1) BUSCO v5.4.0 combined with the eukaryotic core gene database (eukaryota_odb10) was used to evaluate the integrity of the gene region; (2) Merqury was used to evaluate the assembly accuracy and integrity based on k-mer distribution; (3) QUAST was used to statistically analyze the assembly continuity index. Through a hierarchical error correction and ultra-long read priority assembly strategy, a complete draft of the Litopenaeus vannamei genome was finally obtained. The assembly results showed that the total genome size was 2,443,336,735 bp (approximately 2.44 Gb), and the contig N50 reached 15.43 Mb. The BUSCO evaluation results showed that the core gene integrity was over 95%, indicating that the assembly had extremely high continuity and integrity, reaching the level of chromosome-level fragmentation.

[0052] 5. Based on the initial assembly, homology alignment-assisted gap filling and telomere structure optimization were performed using reference genomes from closely related species to further improve the continuity and integrity of the genome. After correction and gap filling, a high-quality reference genome of Litopenaeus vannamei was finally obtained, with a total length of 2.44 Gb and a contig N50 increased to 22.12 Mb. A total of 58 telomere structures were identified, indicating that most chromosomes had achieved telomere-to-telomere coverage. Gap analysis showed that the remaining gaps were mainly concentrated in regions rich in simple repetitive sequences, with the chromosome with the fewest gaps containing only one gap, reflecting that the genome assembly was close to the level of a complete genome.

[0053] 6. The genome integrity was assessed using BUSCO v5.4.0 in conjunction with the eukaryotic core gene database. The results showed a BUSCO integrity of 96.7% at the genome level, indicating that the vast majority of conserved single-copy genes in the genome had been fully assembled, yielding the Litopenaeus vannamei reference genome. Based on the gene set obtained through homology prediction and transcriptome-assisted annotation, its BUSCO integrity reached 97.6% (96.4% single-copy genes and 1.2% repetitive genes), further validating the accuracy and completeness of the gene annotation. Those skilled in the art can easily assemble the Litopenaeus vannamei reference genome using the above steps, resulting in a total genome length of 2.44 Gb, a Contig N50 of 22.12 Mb, and the identification of 58 telomere structures, with a BUSCO integrity of 96.7% at the genome level.

[0054] S2. Sample collection and sequencing, the specific steps are as follows: Four populations of Litopenaeus vannamei were collected, with approximately 1000 shrimp in each population. The body length of each shrimp was measured using a ruler (accurate to 1 mm), and samples of their antennae were collected for genome analysis. The antennae samples were sent to Beijing Novogene Technology Co., Ltd. for genome resequencing. The resequencing data were aligned to the Litopenaeus vannamei reference genome obtained in step S1 above to obtain the reference genome alignment results.

[0055] S3. Genome-wide association analysis, the specific steps of which are as follows: 1. Based on the reference genome alignment results in step S2, GWAS analysis was performed in the four populations using the MLM model of GAPIT software. Covariate correction was performed on the first ten principal components in the age, sex and population structure analysis, and the p-value was used to measure the significance of the association markers.

[0056] The results are as follows Figure 1 As shown, the significance threshold is set to 5 × 10⁻⁶. -8 Based on the p-values ​​associated with meta-GWAS analysis, 18,910,074 SNP loci were identified that were significantly associated with the body length growth trait of Litopenaeus vannamei.

[0057] 2. Meta-analysis was performed on the above GWAS results using the fixed-effects inverse variance weighted model in metal software. The Z-score of each site was obtained, and SNP sites with significant association were selected based on the P-value.

[0058] The formula is as follows: ; ; In the fixed-effects inverse variance weighted model: This represents the effect estimate at a specific locus in the i-th study or population. The standard error of the estimate for this effect reflects the uncertainty of the estimate; the weights constructed from this are... =1 / 2 This indicates the contribution of the study to the pooled analysis; the smaller the standard error, the greater the weight. k is the total number of studies participating in the pooling, and ∑ represents the weighted summation of all studies. The result is obtained through weighting. To estimate the overall effect after merging, This corresponds to the standard error of merging, while = / This is the standardized statistic after pooling. Under the null hypothesis, It approximately follows a standard normal distribution, where Ф is the cumulative distribution function of the standard normal distribution, used to calculate the two-sided significance level. .

[0059] S4. Fine localization of significant SNP sites, the specific steps are as follows: 1. Based on the meta-analysis results obtained in step S3 above, PLINK was first used to perform clumping analysis on significant SNPs (window size: 500 kb; r 2 Threshold: 0.1) to identify mutually independent association signals and determine the lead SNPs and their corresponding candidate genomic regions in each signal. Each clumped associated region is treated as an independent candidate site, and the Z-score and corresponding linkage disequilibrium (LD) matrix of all SNPs within that region are extracted.

[0060] 2. Based on the results obtained in step S4.1 above, the posterior inclusion probability of each SNP as a causal variant is further calculated using CARMA software, and a reliable set is constructed accordingly.

[0061] The formula is as follows: ; in, Here, Z represents the SNP locus, and Z is the Z-score vector from the GWAS summary statistics. For the SNP set of causal variation, SNP sites The posterior ratio, For each SNP site in the causal variation SNP set M. Let be the posterior ratio of each SNP in the causal variation SNP set M.

[0062] 3. Further integrate the results of fine-mapping of loci with the association strength information of meta-GWAS, and extract indicators such as lead SNPs, highest PIP values, and size of the confidence set for comprehensive comparison and summary across regions. Use the genome annotation tool SnpEff to annotate significant SNP loci to determine their location and potential function, and treat genes within 50Kb regions upstream and downstream of significant SNP loci as candidate genes.

[0063] The results are as follows Figure 2 As shown, the posterior inclusion probability of site Chr24_14603628 is 0.99, indicating that this site is highly likely to be the causal variant driving this associated signal. This site is located at position 14603628 on chromosome 24 of the Litopenaeus vannamei reference genome obtained after assembly in step S1 above. The nucleotide flanking sequence of this SNP marker Chr24_14603628 is shown in SEQ ID NO.1.

[0064] Annotated using SNPEff, this site was identified as the TAUD gene (SEQ ID NO.2), located in the upstream 5' regulatory region of the TAUD gene and linked to it. This SNP site influences the expression of the TAUD gene.

[0065] Further genotyping of the SNP marker in SEQ ID NO.1 revealed that the genotypes of the SNP marker Chr24_14603628 are CC, TC, and TT. Specifically, the SNP molecular marker shown in SEQ ID NO.1 has a C or T base at position 504 from the 5' end, classifying it as a T / C type SNP molecular marker. After classifying Litopenaeus vannamei individuals using genotypes, association analysis was performed on the body length and weight of the Litopenaeus vannamei.

[0066] 4. To verify the reliability of the SNP marker Chr24_14603628, one-way ANOVA was used to detect the differences in body length among the three genotypes of Litopenaeus vannamei (main trait verification). T-tests were used to compare body lengths pairwise among different genotypes, i.e., TT vs CC, TC vs TT, and CC vs TC. At the same time, body weight, which is related to body length, was selected as an auxiliary trait to analyze the effect of SNP genotype on body weight (related trait verification).

[0067] The results are shown in Table 3 and Figure 3 As shown, the SNP marker Chr24_14603628 exhibited significant differences in body length and weight between TT and CC types, and between TC and TT types (one-way ANOVA: P-value < 0.05; pairwise t-test: P-value < 0.05). Specifically, the mean body length of TT-type Litopenaeus vannamei was significantly greater than that of TC-type and CC-type Litopenaeus vannamei (P < 0.05), and the mean body length of TC-type Litopenaeus vannamei was significantly greater than that of TC-type Litopenaeus vannamei (P < 0.05).

[0068] Table 3. Correlation analysis between SNP marker Chr24_14603628 (T>C) and body length of Litopenaeus vannamei. ;

[0069] S5. Extraction of Litopenaeus vannamei genomic DNA, the specific steps are as follows: To verify the genotype of significant body length advantage in Litopenaeus vannamei, 15 Litopenaeus vannamei were randomly selected from a 9-month-old population. The shrimp antennae were harvested and ground using liquid nitrogen to obtain shrimp antennae powder from different individuals (hereinafter referred to as antennae powder). Genomic DNA was extracted from the Litopenaeus vannamei using a marine animal genome extraction kit (Tiangen Biotech; all reagents and consumables described below are from this kit). The DNA extraction steps are as follows: 1. Sample pretreatment: Weigh 30 mg of shrimp whisker powder, add 200 μL of GA buffer to a centrifuge tube, mix gently, and shake for 15 seconds to ensure complete infiltration of muscle tissue.

[0070] 2. Proteinase K digestion: Add 20 μL of Proteinase K solution (20 mg / mL) to a centrifuge tube, mix thoroughly, and incubate in a 55°C metal bath until the tissue is completely dissolved.

[0071] 3. Pyrolysis and precipitation: Add 200 μL of GB lysis buffer, mix thoroughly, place in a 70℃ metal bath for 10 minutes, then add 200 μL of anhydrous ethanol and mix well.

[0072] 4. Adsorption column purification: Transfer the mixture to a CB3 adsorption column, place it in a collection tube, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid. Then add 500 μL of GD buffer and 600 μL of PW wash buffer, centrifuging for 30 seconds each time to wash the adsorption column to remove impurities.

[0073] 5. Drying the adsorption column: Place the cleaned adsorption column back into a centrifuge tube, centrifuge at 12,000 rpm for 2 minutes, and then let it stand at room temperature for 2-5 minutes to ensure that the ethanol in the adsorption column is completely evaporated.

[0074] 6. DNA elution: Place the adsorption column into a new centrifuge tube, add 50-200 μL of sterile water to the middle of the adsorption column, let it stand at room temperature for 2 minutes, then centrifuge at 12000 rpm for 2 minutes and collect the eluted DNA solution.

[0075] 7. Quality Inspection and Preservation: A spectrophotometer is used to detect the concentration and purity of the DNA to ensure OD... 260 / OD 280 Between 1.8 and 2.0, OD 260 / OD 230 Greater than 2.0.

[0076] The final concentration of Litopenaeus vannamei genomic DNA obtained was 100 ng / μL. The Litopenaeus vannamei genomic DNA was detected by 1.5% agarose gel electrophoresis (first agarose gel electrophoresis). The remaining Litopenaeus vannamei genomic DNA was stored at -20℃ for later use.

[0077] The results of the first agarose gel electrophoresis are as follows: Figure 4 As shown, M is the marker, 1 is the pure water blank control, and 2-16 are genomic DNA samples of the Litopenaeus vannamei individuals to be tested. All samples are over 5000 bp in length and have good integrity, and can be used for subsequent experiments.

[0078] S6. Primer design and PCR amplification reaction, the specific steps are as follows: 1. Primers were designed using the NCBI primer design module Primer-BLAST to target the SEQ ID NO.1 sequence (hereinafter referred to as the target sequence) containing the SNP molecular marker in Litopenaeus vannamei: Forward primer F: 5'-AATCCGCCACTGAGCCAATTA-3' (SEQ ID NO.3); Reverse primer R: 5'-GAAGGCCGAGTAACCGACTT-3' (SEQ ID NO.4).

[0079] The synthesized SNP molecular marker primers (F and R) were sent to Beijing Qingke Biotechnology Co., Ltd. for primer synthesis, which will be used for subsequent PCR amplification reactions.

[0080] 2. Using the Litopenaeus vannamei genomic DNA obtained in step S5 above as a template, PCR amplification of the target sequence was performed using the synthesized SNP molecular marker primers (SEQ ID NO.3 and SEQ ID NO.4). The specific steps are as follows: (1) The PCR amplification reaction system (25 μL system) was designed as follows: Genomic DNA of Litopenaeus vannamei (100 ng / μL), 1 μL; Forward primer F (10 μM), 1 μL; Reverse primer R (10 μM), 1 μL; 2×Taq Plus Master Mix II (Plus), 12.5 μL; Enzyme-free sterile water, 9.5 μL.

[0081] (2) The PCR amplification reaction procedure is as follows: Pre-denaturation at 95℃ for 3 min; Denaturation at 95℃ for 15 seconds; Anneal at 56℃ for 20 seconds; The denaturation and extension process was performed at 72°C for 40 seconds, with a total of 32 cycles. Extend the heat at 72℃ for 10 minutes. Store at 4°C.

[0082] After the PCR amplification reaction was completed, the PCR amplification products were obtained, and the PCR amplification products were detected by 1.5% agarose gel electrophoresis (second agarose gel electrophoresis).

[0083] The results of the second agarose gel electrophoresis are as follows: Figure 5 As shown, M is the marker; 1 is the blank control; and 2-16 are the target sequences, which have a bright band at 878 bp.

[0084] The verification of S7 and SNP sites follows these steps: 1. The PCR amplification product obtained in step S6 above was sent to Beijing Qingke Biotechnology Co., Ltd. for forward sequencing. The sequencing primer was forward primer F (SEQ ID NO.3): 5'-AATCCGCCACTGAGCCAATTA-3'. For specific primer design and sequencing procedures, please refer to [link to documentation]. Figure 6 ,Depend on Figure 6 It can be seen that the target sequence SEQ ID NO.1 was amplified by PCR using forward primer SEQ ID NO.3 and reverse primer SEQ ID NO.4 to obtain the PCR amplification product. Then, the PCR amplification product was forward sequenced using sequencing primer SEQ ID NO.3 to obtain the sequencing result SEQ ID NO.5 of the SNP site Chr24_14603628.

[0085] 2. Based on the sequencing peak diagram of the PCR amplification products ( Figure 7 Genotyping was performed on the sequencing results of the SNP site Chr24_14603628 (G: SEQ ID NO.5). The sequence length of SEQ ID NO.5 is shorter than that of SEQ ID NO.1, meaning that the molecular marker Chr24_14603628 is located at position 451 from the 5' end as shown in SEQ ID NO.1, which is a T / C type SNP molecular marker. This is equivalent to the molecular marker Chr24_14603628 being located at position 451 from the 5' end as shown in SEQ ID NO.5, which is a T / C type SNP molecular marker.

[0086] If there is only one C base peak at position 451 of SEQ ID NO.5, the genotyping result is CC type; if there is only one T base peak at position 451 of SEQ ID NO.5, the genotyping result is TT type; if there are both C and T base peaks at position 451 of SEQ ID NO.5, the genotyping result is T / C type.

[0087] SNP genotyping results can be used for individual identification of broodstock, population selection, and strain maintenance. Among them, the T genotype is the dominant genotype. In the process of joint selection of body length and weight of Litopenaeus vannamei, male or female individuals with both SNP markers being TT type are retained for breeding and propagation; or individuals with one SNP locus of TT type are selected from the male and female individuals and individuals with another SNP locus of TC type are selected for crossbreeding and propagation. When the stock is available, individuals with both male and female individuals being TC type are selected for crossbreeding, and individuals with the TT genotype are further selected from the offspring.

[0088] Select TT homozygous Litopenaeus vannamei individuals with advantages in both body length and weight, and exclude Litopenaeus vannamei individuals with slow growth in body length and weight as early as possible, thereby increasing the farming yield of Litopenaeus vannamei.

[0089] The verification of the S8 and TAUD genes is carried out through the following steps: Further functional validation of the TAUD gene (SEQ ID NO. 2) annotated with the above Litopenaeus vannamei SNP markers was performed, specifically including the following steps: 1. The specific steps for farming Litopenaeus vannamei are as follows: Litopenaeus vannamei was cultured in a recirculating seawater system at a temperature of 27±1℃. Commercial feed pellets (available commercially) were fed three times daily at a rate of 5% of the shrimp's body weight, with the water changed daily. Seventy Litopenaeus vannamei shrimp, measuring 6.29±0.03 g in weight and 7.18±0.011 cm in length, were selected as experimental subjects; at this stage, the shrimp were in their larval stage.

[0090] 2. RNA interference in Litopenaeus vannamei, the specific steps are as follows: (1) The above 70 cultured Litopenaeus vannamei shrimp were divided into two equal parts, and a negative control group (siNC) and an experimental group (siTAUD) were set up, with 35 shrimp in each group.

[0091] (2) Using the siDireTC v2.1 tool, siRNA sequence pairs targeting the TAUD gene (SE QID NO.2) were designed, where, Negative control group primer design: siNC-F: 5'-ATGGGCTGGTGGAAGAAG-3' (SEQ ID NO. 6); siNC-R: 5'-CTGAAGGGGAAGACGGAG-3' (SEQ ID NO. 7); Experimental primer design: siTAUD-F: 5'-TATGCATGGAAAGGAAGGTGTCATAAGGAT-3' (SEQ ID NO. 8); siTAUD-R: 5'-CTGTTTCAGAGAGCAGATTGACATCAAC-3' (SEQ ID NO. 9); The above four primers (SEQ ID NO.6~SEQ ID NO.9) were delivered to Beijing Qingke Biotechnology Co., Ltd. for primer sequence synthesis.

[0092] (3) The primers synthesized from the above SEQ ID NO.6~SEQ ID NO.9 sequences were centrifuged at 12000 r / min for 1 min, and then dissolved in enzyme-free sterile water. After that, dsRNA was synthesized in vitro.

[0093] Negative control group (siNC): Each Litopenaeus vannamei was injected with EGFP-dsRNA dissolved in enzyme-free sterile water at a dose of 2 μg / g / shrimp (i.e., each Litopenaeus vannamei was injected with 2 μg per g of body weight, and so on, increasing the injection dose according to the body weight of each shrimp). Experimental group (siTAUD): Each Litopenaeus vannamei was injected with siTAUD-dsRNA dissolved in enzyme-free sterile water at a dose of 2 μg / g / shrimp (same as the negative control group); injections were given every 4 days, targeting the middle of the second to third abdominal segments. The experiment lasted for 22 days. After the experiment, abdominal nerve samples from all Litopenaeus vannamei shrimp were collected and stored at -80℃. The body length of each Litopenaeus vannamei shrimp was measured (accurate to mm) before each injection and before sample collection.

[0094] Measurement results as follows Figure 8 As shown, the body length growth of Litopenaeus vannamei in the experimental group was significantly lower than that in the negative control group (siNC). The body length of Litopenaeus vannamei in the experimental group increased by about 0.8 cm, while the body length of the negative control group (siNC) increased by about 2.6 cm. This indicates that interfering with the expression of the TAUD gene can significantly inhibit the growth rate of Litopenaeus vannamei body length.

[0095] 3. The relative expression level of the TAUD gene in Litopenaeus vannamei was determined, and the specific steps are as follows: (1) Extraction of total RNA from Litopenaeus vannamei Twenty-four shrimp were randomly selected from the aforementioned 70 Litopenaeus vannamei. Total RNA was extracted from the shrimp intestines using the Trizol method. The concentration and quality of the extracted RNA were evaluated by Nanodrop 2000 and 1.5% agarose gel electrophoresis. The specific experimental steps for RNA extraction using the Trizol method are as follows: 1) After cleaning the pipette tips, eight-piece pipettes, grinding magnetic beads, scissors, tweezers and other experimental instruments, put them in an autoclave for sterilization, and then put them in an oven for use. 2) Cut 50 μg of Litopenaeus vannamei intestine and place it in a 1.5 mL enzyme-free centrifuge tube, then add 500 μL of Trizol to soak the tissue; 3) Add 3-5 grinding magnetic beads to the centrifuge tube, place the centrifuge tube in a tissue homogenizer for grinding, and grind under the conditions of 70 Hz, 4℃, for 3 min. 4) After grinding the tissue, add 500 μL of Trizol to the centrifuge tube, mix well, and place on ice for 5 min; 5) Centrifuge at 12000 rpm and 4℃ for 5 min; transfer the supernatant to a new centrifuge tube, add about 200 μL of chloroform, shake vigorously for about 15 s to mix, and place on ice for 15 min. 6) Centrifuge at 12000 rpm and 4℃ for 15 min; transfer the supernatant to another centrifuge tube, add an equal amount of isopropanol pre-cooled at 20℃, mix gently, and let stand at 20℃ for 2 h. 7) Centrifuge at 1200 rpm and 4℃ for 10 min; discard the supernatant, add 1 mL of pre-cooled 75% ethanol, gently shake, and suspend the precipitate; 8) Centrifuge at 10,000 rpm, 4℃ for 5 min; discard the supernatant, add 1 mL of pre-cooled anhydrous ethanol, gently shake to suspend the precipitate; 9) Centrifuge at 10,000 rpm, 4℃ for 5 min; discard the supernatant, open the centrifuge tube cap and air dry in a clean bench for 5-10 min, then add about 50 μL of DEPC water to dissolve the precipitate; 10) Take 1 µL of RNA sample and determine its concentration and purity using a Nanodrop 2000 micro spectrophotometer; 11) Take 1 µg of RNA sample and use 1.5% lipoglycol gel electrophoresis to check its integrity and observe whether there are obvious 28S and 18S rRNA bands; store the qualified RNA sample in a -80℃ freezer.

[0096] The results of total RNA extraction from shrimp intestines after RNA interference are as follows: Figure 9 As shown, M is the marker, and 1-24 are the total RNA from the shrimp intestine after RNA interference. The quality of the extracted total RNA is good and can be used for subsequent experiments.

[0097] (2) Reverse transcription Using the total RNA extracted from Litopenaeus vannamei in step (1) above as a template, reverse transcription was performed using the All In One 5X RTMasterMix kit (all reagents are included in the kit).

[0098] The reverse transcription system was: 4 μL of 5X All In One RT MasterMix, 1 μg of total RNA, and brought to a final volume of 20 μL with RNase-free water. The reverse transcription program was as follows: 37℃ for 15 min, 60℃ for 10 min, 95℃ for 3 min, and stored at 4℃ to obtain cDNA from Litopenaeus vannamei.

[0099] (3) qRT-PCR Using the cDNA of Litopenaeus vannamei obtained in step (2) above, diluted 5-fold as a template, and EF1A as an internal reference gene, specific primers for the TAUD gene and the internal reference gene were designed using NCBI. Upstream primer EF1A-F: 5'-ATGGGCTGGTGGAAGAAG-3' (SEQ ID NO.10); Downstream primer EF1A-R: 5'-CTGAAGGGGAAGACGGAG-3' (SEQ ID NO.11); Upstream primer TAUD-F: 5'-AGTCCTTGAATGCTTGGGTT-3' (SEQ ID NO.12); Downstream primer TAUD-R: 5'-CCAGCCTCTATTTTAGGTTTCTC-3' (SEQ ID NO.13).

[0100] The expression level of the target gene was detected by qRT-PCR using the BlasTaq 2X qPCR MasterMi kit. The qRT-PCR reaction system was as follows: 2 μL of cDNA from Litopenaeus vannamei, 10 μL BlasTaq 2X qPCR MasterMix, 0.5 μL upstream primer (10 μM); 0.5 μL of downstream primer (10 μM), 7 μL DEPC water.

[0101] Each sample was configured with 3 biological replicates, and each biological replicate was configured with 4 technical replicates. The samples were loaded into 96-well plates and centrifuged at 3000 r / min for 1 min. qRT-PCR was performed using a real-time quantitative PCR instrument. The qRT-PCR reaction program was as follows: pre-denaturation at 95℃ for 30 s, denaturation at 95℃ for 10 s, annealing and extension at 60℃ for 30 s, for a total of 40 cycles from denaturation to annealing and extension.

[0102] The specificity of the primers was verified by melting curve analysis, using 2 -ΔΔt The relative expression level of the target gene can be calculated.

[0103] qRT-PCR results are as follows Figure 10 As shown, the relative expression level of the TAUD gene in the intestinal tissue of Litopenaeus vannamei was lower in the experimental group than in the negative control group. The results indicate that siTAUD effectively inhibited the expression of the TAUD gene and significantly affected the growth traits of Litopenaeus vannamei.

[0104] In conclusion, the TAUD gene in Litopenaeus vannamei can regulate its growth rate. Brief treatment of larval Litopenaeus vannamei using RNA interference revealed that siTAUD significantly inhibited TAUD gene expression in the shrimp intestine, reducing wasted TAUD gene expression during the larval stage and minimizing growth rate differences among individuals within the population. After restoring TAUD gene expression, overexpression of the TAUD gene can be used to accelerate individual growth, improve farming efficiency, and increase economic value.

[0105] Comparative Example 1 The difference between this comparative example and the embodiment is that the primers used to detect SNP markers are: LvSRC-F: 5'-CGGTGTTCATAAAGAGGATAC-3' (SEQ ID NO. 14); LvSRC-R: 5'-TCACCACTCCAACAGACGAAGG-3' (SEQ ID NO. 15); The primers used in this comparative example are the publicly available SNP sites for detecting the rapid growth rate of Litopenaeus vannamei reported in the literature.

[0106] The results showed that the primers provided in this comparative example could not detect the SNP markers in this example, while the primer set provided by this invention could be used to perform PCR reaction to quickly detect the SNP molecular markers in Example 1.

[0107] Comparative Example 2 The difference between this comparative example and the embodiment is that this comparative example uses the RNA interference primers described in patent document CN119979733A to interfere with RNA in Litopenaeus vannamei via in vitro injection. The specific RNA interference primers are as follows: siPDE11A-F: 5'-CCGCATCATGACACATACA-3' (SEQ ID NO. 16); siPDE11A-R: 5'-TGTATGTGTCATGATGCGG-3' (SEQ ID NO. 17).

[0108] The results showed that although the RNA interference primers in this comparative example could inhibit the body length growth of Litopenaeus vannamei individuals, the effect was still significantly lower than that of the RNA interference primers in this application. Furthermore, the RNA interference primers in this comparative example did not affect the expression of the TAUD gene.

[0109] Therefore, it can be concluded that this invention locates a body length-related SNP molecular marker site in the 5' regulatory region upstream of the TAUD gene in Litopenaeus vannamei through genome-wide association analysis. The sequence of the Chr24_14603628 molecular marker is shown in SEQ ID NO.1, and the SNP site is located at position 504 from the 5' end of the sequence shown in SEQ ID NO.1, exhibiting a T / C polymorphism. Primers SEQ ID NO.3 and SEQ ID NO.4, designed based on the sequence in SEQ ID NO.1, can detect the Chr24_14603628 molecular marker, and these primers can also be prepared into a kit. Simultaneously, the siRNA sequence targeting the TAUDE gene, when injected in vitro into Litopenaeus vannamei, significantly inhibits the body length growth rate of the shrimp. Subsequent overexpression of the TAUD gene in Litopenaeus vannamei can achieve rapid body length growth. Therefore, the SNP molecular marker Chr24_14603628 provided by this invention can be used to assist in the selection of fast-growing parents. By using the TT or TC genotype in this SNP molecular marker to select parents, the growth rate of the breeding population can be improved, providing an innovative tool for molecular breeding of Litopenaeus vannamei, shortening the breeding cycle, and contributing to industrial development and efficiency improvement.

[0110] In the foregoing description of exemplary embodiments / specific implementations of this patent, various features of this patent are sometimes combined in a single embodiment / specific implementation or its figures and description, with the aim of simplifying the disclosure and aiding in the understanding of one or more of the various aspects of the invention. However, unless expressly stated otherwise or in obvious technical contradiction or exclusion, the descriptive method of this patent should not be construed as reflecting an intention that the claimed features of the invention are more than expressly stated in each claim. Rather, the inventive aspect reflected in the claims lies in not all the features of a single foregoing disclosed embodiment / specific implementation. Therefore, the claims following the detailed description are hereby expressly incorporated into this detailed description, each claim existing independently as a separate embodiment / specific implementation of this patent.

[0111] The terms and expressions used in this specification are for illustrative purposes and not for limitation. Their use is not intended to exclude any equivalents of the shown and described features or portions thereof, but rather to facilitate the understanding that various modifications may be possible within the scope of this patent claim. Therefore, it should be understood that while this patent has been specifically disclosed through preferred embodiments, exemplary embodiments, and optional features, variations or modifications of the concepts disclosed herein may be adopted by those skilled in the art, and such variations and modifications are therefore considered to be within the scope of this patent as defined by the appended claims. The specific embodiments given in this specification are examples of useful embodiments of this patent, and it will be apparent to those skilled in the art that this patent can be implemented using many variations of the devices, device components, and method steps disclosed herein.

[0112] The foregoing description of specific embodiments fully discloses the general features of this patent, enabling others to easily modify and / or adapt such embodiments for various applications by applying knowledge within the scope of the art, without excessive experimentation or deviation from the general concept of this patent. Therefore, based on the teachings and guidance provided herein, it is intended that such modifications and alterations be included within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive purposes and not intended to be limiting; thus, the wording or terminology in this specification will be interpreted by those skilled in the art based on the foregoing teachings and guidance.

[0113] Furthermore, the scope of this patent should not be limited to any of the exemplary embodiments described above, but only to the appended claims and their equivalents.

Claims

1. An application of SNP markers in the breeding of body length and weight of Litopenaeus vannamei, characterized in that, The sequence of the SNP marker is shown in SEQ ID NO.

1. The SNP site is located at position 504 from the 5' end of the sequence shown in SEQ ID NO.

1. The polymorphism of the SNP marker is T / C type. The SNP marker can simultaneously filter the body length and weight of the Litopenaeus vannamei.

2. The application of the SNP marker according to claim 1 in the breeding of body length and weight of Litopenaeus vannamei, characterized in that, The SNP marker is located in the upstream 5' regulatory region of the TAUD gene, the sequence of which is shown in SEQ ID NO.

2.

3. The application of a primer pair for detecting SNP markers in the breeding of body length and weight of Litopenaeus vannamei, characterized in that, The base sequence of the primer pair is: Forward primer F: 5'-AATCCGCCACTGAGCCAATTA-3'; Reverse primer R: 5'-GAAGGCCGAGTAACCGACTT-3'; The sequence of the SNP marker is shown in SEQ ID NO.

1. The SNP site is located at position 504 from the 5' end of the sequence shown in SEQ ID NO.

1. The polymorphism of the SNP marker is T / C type.

4. The application of an SNP marker kit in the breeding of body length and weight of Litopenaeus vannamei, characterized in that, The kit includes primer pairs, the base sequences of which are: Forward primer F: 5'-AATCCGCCACTGAGCCAATTA-3'; Reverse primer R: 5'-GAAGGCCGAGTAACCGACTT-3'; The sequence of the SNP marker is shown in SEQ ID NO.

1. The SNP site is located at position 504 from the 5' end of the sequence shown in SEQ ID NO.

1. The polymorphism of the SNP marker is T / C type.

5. A method for breeding Litopenaeus vannamei, characterized in that, Includes the following steps: Step S1: Detect the SNP molecular markers of Litopenaeus vannamei to obtain the genotype of the SNP site; the sequence of the SNP marker is shown in SEQ ID NO.1; the SNP site is located at position 504 from the 5' end of the sequence shown in SEQ ID NO.1; the polymorphism of the SNP molecular marker is T / C type; Step S2: Simultaneously screen the body length and weight of the Litopenaeus vannamei based on the genotype of the SNP locus; the genotype of the SNP locus is TT, TC, or CC; Step S3: Use the Litopenaeus vannamei male and female parents with the TT genotype at the SNP locus for crossbreeding.

6. The method for breeding Litopenaeus vannamei according to claim 5, characterized in that, Step S1 includes: Step S1-1: Extract the genomic DNA of the Litopenaeus vannamei; Step S1-2: Using the genomic DNA of the Litopenaeus vannamei as a template, PCR amplification was performed using the following primer pairs to obtain the PCR amplification product; Forward primer F: 5'-AATCCGCCACTGAGCCAATTA-3'; Reverse primer R: 5'-GAAGGCCGAGTAACCGACTT-3'; Steps S1-3: Sequencing is performed using the PCR amplification product to obtain the genotype of the SNP site. The genotype of the SNP site is the T / C type at position 504 from the 5' end of the sequence shown in SEQ ID NO.

1.

7. The method for breeding Litopenaeus vannamei according to claim 6, characterized in that, In steps S1-2, The PCR amplification system is a 25 μL amplification system; the 25 μL amplification system includes: 1 μL of Litopenaeus vannamei genomic DNA at a concentration of 100 ng / μL; 1 μL of forward primer F at a concentration of 10 μM; 1 μL of reverse primer R at a concentration of 10 μM; 12.5 μL of 2×Taq Plus Master Mix II; and 9.5 μL of enzyme-free sterile water; The PCR amplification program consists of the following steps: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at 56℃ for 20 s, extension at 72℃ for 40 s, for a total of 32 cycles from denaturation to extension, followed by a full extension at 72℃ for 10 min.

8. The method for breeding Litopenaeus vannamei according to claim 5, characterized in that, In step S2 The criteria for screening the genotypes of the SNP loci are as follows: the body length and weight of Litopenaeus vannamei individuals with the TT genotype of the SNP loci > the body length and weight of Litopenaeus vannamei individuals with the TC genotype of the SNP loci > the body length and weight of Litopenaeus vannamei individuals with the CC genotype of the SNP loci.

9. The method for breeding Litopenaeus vannamei according to claim 5, characterized in that, Specifically, step S3 involves retaining Litopenaeus vannamei with the TT genotype at the SNP locus and breeding them by pairing male and female Litopenaeus vannamei with the TT genotype.

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