SNP molecular marker for growth traits of litopenaeus vannamei, detection primer, kit and application thereof

Abstract

The application discloses a SNP molecular marker of Litopenaeus vannamei growth traits, a detection primer, a kit and application thereof. The SNP molecular marker is shown as SEQ ID NO. 1, and the polymorphism of the base at the 497th position from the 5' end of SEQ ID NO. 1 is A / G; the SNP molecular marker can simultaneously screen body length and weight to carry out Litopenaeus vannamei breeding, and the SNP molecular marker can also be detected by using a primer designed according to the sequence of SEQ ID NO. 1 or prepared into a kit. Meanwhile, the AA genotype of the SNP molecular marker Chr25_11409188 provided in the application can be used to select Litopenaeus vannamei parents, further improve the growth speed of the body length and weight of a breeding population, provide an innovative tool for molecular breeding of Litopenaeus vannamei, shorten a breeding cycle, and help industrial development and efficiency improvement.

Description

Technical Field

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

[0002] Litopenaeus vannamei, also known as the Pacific white shrimp, belongs to the phylum Arthropoda, class Crustaeea, order Decapoda, family Penaeidae, and genus Penaeus. It is a pillar species in the global aquaculture industry, boasting the highest production volume and being one of the highest-value aquaculture species. Litopenaeus vannamei is widely farmed due to its rapid growth, strong environmental adaptability, and high economic value. However, significant differences exist in the growth performance of Litopenaeus vannamei during farming. Shrimp growth traits are primarily influenced by various genetic and environmental factors. Among these, body length and weight are crucial indicators of economic value; shorter body length or lower weight significantly impacts farming efficiency and economic returns.

[0003] Molecular breeding of Litopenaeus vannamei mainly relies on traditional breeding selection methods. Although these methods can improve growth traits to some extent, they have the following drawbacks: (1) Long breeding cycle and low efficiency: Traditional breeding methods select individuals with better performance for reproduction. However, due to the strong quantitative and environmental influence of growth traits, the breeding cycle is long and the efficiency is low, making it difficult to meet the growing market demand; (2) Lack of precise molecular markers: Although some genomic technologies (such as genome selection and molecular marker-assisted selection) have been applied to shrimp breeding, there is still a lack of efficient molecular markers for growth traits. Existing markers are often too generalized and cannot accurately identify genetic factors that are highly related to growth traits, which limits the efficiency of genetic improvement; (3) Difficulty in achieving precision in genetic improvement: Due to the complex genetic basis of growth traits in Litopenaeus vannamei, traditional phenotypic selection methods are often greatly affected by environmental factors, making it difficult to achieve precise screening and transmission of superior growth traits.

[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 molecular 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] Therefore, the development of single SNP molecular markers for simultaneous screening of body length and weight growth traits in Litopenaeus vannamei is of great significance for the breeding of new varieties of Litopenaeus vannamei. It provides an innovative tool for molecular breeding of Litopenaeus vannamei, adds screening criteria to the traditional single SNP molecular marker screening of a single growth trait, makes SNP molecular marker screening more accurate, further shortens the breeding cycle, and helps the industry develop and improve efficiency. Summary of the Invention

[0006] The purpose of this invention is to develop SNP molecular markers, detection primers, kits, and applications for growth traits in Litopenaeus vannamei. This allows for the simultaneous screening of body length and weight growth traits in Litopenaeus vannamei using a single SNP molecular marker. This adds a screening criterion to the traditional method of screening a single growth trait using a single SNP molecular marker, making SNP molecular marker screening more precise, accelerating the breeding and genetic improvement of fast-growing Litopenaeus vannamei varieties, and improving breeding efficiency and economic benefits.

[0007] To solve this technical problem: This patent provides an SNP molecular marker, as shown in SEQ ID NO.1, where the polymorphism of the 497th base from the 5' end is A / G.

[0008] Another aspect of this patent provides the application of a reagent for detecting SNP molecular markers in the breeding of Litopenaeus vannamei. The SNP molecular marker is shown in SEQ ID NO.1, and the polymorphism of the 497th base from the 5' end of SEQ ID NO.1 is A / G. The breeding program was conducted for long-bodied and heavy-bodied Litopenaeus vannamei individuals. The body length and weight of Litopenaeus vannamei individuals with the AA genotype of the SNP molecular marker were greater than those with the AG genotype of the SNP molecular marker, which in turn were greater than those with the GG genotype of the SNP molecular marker.

[0009] Furthermore, the reagent is a primer pair, and the nucleotide sequence of the primer pair is as follows: Forward primer F: 5'-GTCGAGAAGAGCACCGGTAA-3'; Reverse primer R: 5'-GCAAGCCTGTTACTTCGCAA-3'.

[0010] Another aspect of this patent provides the application of an SNP molecular marker kit in the breeding of Litopenaeus vannamei, characterized in that the kit includes primer pairs, the nucleotide sequences of which are: Forward primer F: 5'-GTCGAGAAGAGCACCGGTAA-3'; Reverse primer R: 5'-GCAAGCCTGTTACTTCGCAA-3'; The SNP molecular marker is shown in SEQ ID NO.1. The polymorphism of the 497th base from the 5' end in SEQ ID NO.1 is A / G. The breeding program was conducted for long-bodied and heavy-bodied Litopenaeus vannamei individuals. The body length and weight of Litopenaeus vannamei individuals with the AA genotype of the SNP molecular marker were greater than those with the AG genotype of the SNP molecular marker, which in turn were greater than those with the GG genotype of the SNP molecular marker.

[0011] 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 genotypes of the SNP molecular markers; the SNP molecular markers are shown in SEQ ID NO.1, and the polymorphism of the 497th base from the 5' end of SEQ ID NO.1 is A / G; Step S2: Screening is performed based on the genotype of the SNP molecular marker, which is AA, AG, or GG; the screening is based on the body length and weight of Litopenaeus vannamei. Step S3: Breed Litopenaeus vannamei with the AA genotype using SNP molecular markers to breed long-bodied and heavy-bodied individuals.

[0012] Furthermore, step S1 includes: Step S1-1: Extract genomic DNA from Litopenaeus vannamei; Step S1-2: Using Litopenaeus vannamei genomic DNA as a template, PCR amplification was performed using primer pairs to obtain PCR amplification products; Steps S1-3: Sequencing the PCR amplification products to obtain the genotypes of the SNP molecular markers.

[0013] Furthermore, in steps S1-2, the nucleotide sequences of the primer pairs are as follows: Forward primer F: 5'-GTCGAGAAGAGCACCGGTAA-3'; Reverse primer R: 5'-GCAAGCCTGTTACTTCGCAA-3'.

[0014] Furthermore, 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.

[0015] Furthermore, the PCR amplification program includes the following steps: pre-denaturation at 95℃ for 2 min, denaturation at 98℃ for 10 s, annealing at 59℃ for 30 s, extension at 68℃ for 1 s, for a total of 30 cycles from denaturation to extension, followed by a full extension at 68℃ for 5 min.

[0016] Furthermore, in step S2, the body length and weight of Litopenaeus vannamei individuals with the SNP molecular marker genotype AA are greater than those with the SNP molecular marker genotype AG, which are greater than those with the SNP molecular marker genotype GG.

[0017] Furthermore, step S3 specifically involves: retaining Litopenaeus vannamei genotype AA with SNP molecular markers, and breeding by pairing male and female Litopenaeus vannamei genotype AA for reproduction.

[0018] Compared with existing technologies, it has the following beneficial technical effects: This application provides a SNP molecular marker for the growth traits of Litopenaeus vannamei, detection primers, a kit, and their applications. The SNP molecular marker is shown in SEQ ID NO.1, where the polymorphism at position 497 from the 5' end is A / G. This SNP molecular marker can simultaneously screen for body length and weight for Litopenaeus vannamei breeding. Primers designed based on the sequence of SEQ ID NO.1 or kits prepared accordingly can also be used to detect this SNP molecular marker. Furthermore, the AA genotype of the SNP molecular marker Chr25_11409188 provided in this application can be used to select Litopenaeus vannamei parents, further improving the growth rate of body length and weight in the breeding population. This provides an innovative tool for molecular breeding of Litopenaeus vannamei, shortens the breeding cycle, and contributes to industrial development and efficiency improvement. Attached Figure Description

[0019] 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.

[0020] 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 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 GG type represented in red, AG type in green, and AA 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 3 The image shows agarose gel electrophoresis of partial Litopenaeus vannamei genomic DNA in the example (where M is the marker; 1 is the blank control; and 2-16 are the target genes, all exceeding 5000 bp in length and with good integrity). Figure 4 The image shows agarose gel electrophoresis images of some Litopenaeus vannamei PCR amplification products in the example (where M is the marker, and 1-4 are all PCR products of the target sequence, with a length of 436 bp). Figure 5 This is a flowchart illustrating the sequence sequencing process in the example. Figure 6 The image shows the Blasten results of the simplified reference sequence SEQ ID NO.5 and the SNP molecular marker sequence SEQ ID NO.1 in the example on NCBI (where Query is the sequence SEQ ID NO.1, from 462-616 bp; Sbjct is SEQ ID NO.5, from 1-155 bp). Figure 7 The image shows the alignment analysis results of the Litopenaeus vannamei PCR amplification product sequence SEQ ID NO.4 and the abbreviated reference sequence SEQ ID NO.5 using APE (where the first and second rows are sequence SEQ ID NO.5, from 1 to 155 bp; the third row is sequence SEQ ID NO.4, from 1 to 436 bp; the bases of the SNP molecular marker sites are marked with black boxes, i.e., Chr25_11409188). Figure 8 The image shows the sequencing results of the PCR amplification products of the Litopenaeus vannamei sample in the example (where the bases of the target SNP molecular marker sites are marked with black borders, i.e., Chr25_11409188). Detailed Implementation

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0026] Unless otherwise specified, all the technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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).

[0031] Unless otherwise specified, percentages (%) in this document refer to percentages by mass relative to the composition.

[0032] Unless otherwise stated herein, the sum of the contents of the components in the composition is 100%.

[0033] 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.

[0034] Unless otherwise specified, the term "a" as used in this specification means "at least one".

[0035] 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.

[0036] 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.

[0037] 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.

[0038] (1) Source of sample materials In this embodiment, Litopenaeus vannamei was purchased from Hainan Renhai Aquatic Products Technology Co., Ltd. (Wenchang City, Hainan Province).

[0039] In this embodiment, primer synthesis was performed by Beijing Qingke Biotechnology Co., Ltd., and sequencing was performed by Beijing Novogene Technology Co., Ltd.

[0040] (2) Sources of reagents and consumables Table 1. Sources and Product Codes of Reagents and Consumables Required for the Experiment ;

[0041] (3) Source of instruments and equipment Table 2. Sources and Models of Instruments and Equipment Required for the Experiment ;

[0042] Example This patent provides an SNP molecular marker, detection primers, kit, and application for the growth traits of Litopenaeus vannamei, including the following steps: S1. Sample collection and sequencing, the specific steps are as follows: Four different populations of Litopenaeus vannamei were collected, with 1000 individuals from each population. The body length of each Litopenaeus vannamei was measured (accurate to 1 mm), and a sample of the antennae of each Litopenaeus vannamei was collected and sent to Beijing Novogene Technology Co., Ltd. for genome resequencing. The genome resequencing data was aligned with the official Litopenaeus vannamei reference genome (GCA_042767895.1) from NCBI to obtain the reference genome alignment results.

[0043] S2. Genome-wide association analysis, the specific steps of which are as follows: 1. Based on the reference genome alignment results in step S1 above, GWAS analysis was performed in four different 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.

[0044] The results are as follows Figure 1 As shown, the Bonferroni significance threshold was set to 5.29 × 10⁻⁶. -8 Significant SNP clusters on chromosome 25 were screened, and the SNP with the smallest P-value, 25_11409188, was extracted from the cluster. This site is located at position 11409189 on chromosome 25 of the Litopenaeus vannamei reference genome. The nucleotide flanking sequence of this SNP molecular marker Chr25_11409188 is shown in SEQ ID NO.1, which is 909 bp in length.

[0045] Further genotyping of the SNP molecular marker in SEQ ID NO.1 revealed that the genotypes of the SNP molecular marker Chr25_11409188 are AA, AG, and GG. Specifically, the SNP molecular marker shown in SEQ ID NO.1 has an A or G base at position 497 from the 5' end, classifying it as an A / G type SNP molecular marker (which can also be understood as the polymorphism of this SNP molecular marker being A / G). Further classification of Litopenaeus vannamei individuals based on genotype led to an association analysis of the body length of the Litopenaeus vannamei.

[0046] 2. To verify the reliability of the SNP molecular marker Chr25_11409188, a T-test was used to compare body lengths between different genotypes, namely AA vs GG, AA vs AG, and AG vs GG. At the same time, body weight, which is related to body length, was selected as an auxiliary trait to analyze the effect of different genotypes of the SNP molecular marker on body weight (related trait verification).

[0047] The results are shown in Table 3 and Figure 2As shown, the SNP molecular marker Chr25_11409188 exhibited significant differences in body length and weight between AA and GG individuals, and between AG and GG individuals (one-way ANOVA: P-value < 0.05 was considered significant; pairwise t-tests: P-value < 0.05 was considered significant). Specifically, the average body length and weight of AA-type Litopenaeus vannamei individuals were significantly greater than those of GG-type individuals (P < 0.05), and the average body length and weight of AG-type Litopenaeus vannamei individuals were also significantly greater than those of GG-type individuals (P < 0.05).

[0048] Table 3. Correlation analysis of Chr25_11409188 A>G with body length and weight of Litopenaeus vannamei. ;

[0049] S3. 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] The results of the first agarose gel electrophoresis are as follows: Figure 3 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.

[0058] S4. 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'-GTCGAGAAGAGCACCGGTAA-3' (SEQ ID NO.2); Reverse primer R: 5'-GCAAGCCTGTTACTTCGCAA-3' (SEQ ID NO.3).

[0059] 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.

[0060] 2. Using the Litopenaeus vannamei genomic DNA obtained in step S3 above as a template, PCR amplification of the target sequence was performed using the synthesized SNP molecular marker primers (SEQ ID NO.2 and SEQ ID NO.3). 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.

[0061] (2) The PCR amplification reaction procedure is as follows: Pre-denaturation at 95℃ for 2 min; Denaturation at 98℃ for 10 seconds; Anneal at 59℃ for 30 seconds; The denaturation and extension process was performed at 68°C for 1 second, with a total of 32 cycles. Extend the heat at 68℃ for 55 minutes. Store at 4℃.

[0062] 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).

[0063] The results of the second agarose gel electrophoresis are as follows: Figure 4 As shown, M is the marker; 1 is the blank control; and 2-16 are the target sequences, which have a bright band at around 500 bp.

[0064] The verification of S5 and SNP molecular markers is carried out through the following steps: 1. The PCR amplification product obtained in step S4 above was sent to Beijing Qingke Biotechnology Co., Ltd. for reverse sequencing. The sequencing primer was reverse primer R (SEQ ID NO.3): 5'-GCAAGCCTGTTACTTCGCAA-3'. For specific primer design and sequencing procedures, please refer to [link to documentation]. Figure 5 ,Depend on Figure 5 It can be seen that, firstly, the target sequence SEQ ID NO.1 is amplified by PCR using the forward primer SEQ ID NO.2 and the reverse primer SEQ ID NO.3 to obtain the PCR amplification product. Then, the PCR amplification product is reverse sequenced using the reverse primer SEQ ID NO.3. The original sequence obtained by sequencing is then subjected to reverse complementation to finally obtain the forward-readable positive strand. The sequence of the positive strand is shown in SEQ ID NO.4, which is 436 bp in length.

[0065] As is well known to those skilled in the art, reverse sequencing is a sequencing method that corresponds to forward sequencing. Reverse sequencing sequences DNA from the 3' end to the 5' end, with designed primers binding to the 5' end of the antisense strand. Reverse sequencing is typically used to confirm the correctness of a sequence.

[0066] 2. Due to the presence of dense polyT and dinucleotide repeat structures such as TA, GT, and AC (also known as simple repeat sequences) in the upstream neighboring region of the SNP molecular marker (referring to the 340-460 bp position of SEQ ID NO.1), the accuracy and peak quality of Sanger sequencing are severely affected. Moreover, as is well known to those skilled in the art, such simple repeat sequences cannot be avoided by optimizing primer design. This application has also attempted to optimize primer design, but it is still impossible to avoid the simple repeat sequences appearing in the PCR amplification products during sequencing.

[0067] Therefore, this application bypasses the upstream simple repetitive sequence of SEQ ID NO.1 by reverse sequencing, and at the same time prunes the interfering sequence to obtain a simplified reference sequence, as shown in SEQ ID NO.5 (marked as SR in the sequence listing), with a length of 155 bp.

[0068] This application further utilizes the Blastn tool from the NCBI official website to perform comparative analysis on SEQ ID NO.5 and SEQ ID NO.1, and the results are as follows: Figure 6 As shown, SEQ ID NO.1 completely covers SEQ ID NO.5, with a similarity of 100%, indicating that the simplified reference sequence SEQ ID NO.5 is the sequence on the SNP molecular marker SEQ ID NO.1 of this application.

[0069] This application further utilizes APE (A Plasmid Editor, sequence analysis software) to compare SEQ ID NO.4 and SEQ ID NO.5. The results are as follows: Figure 7 As shown, the first line is the simplified reference sequence SEQ ID NO.5, the second line is the positive strand of the PCR amplification product SEQ ID NO.4, and finally the 285 bp position on SEQ ID NO.4 is located at the 36 bp position on SEQ ID NO.5. Those skilled in the art can then... Figure 6 It can be directly and without doubt that the 36th bp position of SEQ ID NO.5 is completely identical to the 497th position of SEQ ID NO.1 above, and the 285th bp position on the positive strand of SEQ ID NO.4 of the PCR amplification product is the base of the SNP molecular marker site in SEQ ID NO.1 of this application.

[0070] This indicates that SEQ ID NO.4 belongs to the same SNP molecular marker as SEQ ID NO.5 and SEQ ID NO.1. That is, those skilled in the art can use the primer pair of SEQ ID NO.2 and SEQ ID NO.3 to detect the 497th base on SEQ ID NO.1 and verify the effect of this application in simultaneously screening the dominant genotypes of the two growth traits of Litopenaeus vannamei: body length and weight.

[0071] 3. Genotyping of the PCR amplification product sequencing results (G: SEQ ID NO.4) based on the reverse sequencing peak diagram of the PCR amplification product in step S51 above.

[0072] The results are as follows Figure 8 As shown, the 285th base in SEQ ID NO.4 is either A or G, indicating an A / G type SNP molecular marker (i.e., the polymorphism of this SNP molecular marker is A / G). If there is only one G base peak at position 285 of SEQ ID NO.4, the genotyping result is GG type; if there is only one A base peak at position 285 of SEQ ID NO.4, the genotyping result is AA type; if there are both G and A base peaks at position 285 of SEQ ID NO.4, the genotyping result is AG type.

[0073] SNP genotyping results can be used for individual identification, population selection, and strain maintenance of Litopenaeus vannamei broodstock. Among them, the A genotype is the dominant genotype. In the process of joint selection of body length and weight of Litopenaeus vannamei broodstock, individuals of the male or female line with both SNP molecular markers of AA type are retained for breeding and propagation; or individuals of the male or female line with one SNP molecular marker locus of AA type are selected and mated with individuals of the other SNP molecular marker locus of AG type for propagation; or individuals of the male and female line with both SNP molecular markers of AG type are selected and mated, and Litopenaeus vannamei broodstock individuals with AA genotype are further selected from the offspring.

[0074] Select AA 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.

[0075] Comparative Example The difference between this comparative example and the embodiment is that the SNP molecular markers and primers disclosed in CN121852568A were used to screen for body length and weight of Litopenaeus vannamei: Forward primer Fw: 5'-AATCCGCCACTGAGCCAATTA-3' (SEQ ID NO.6); Reverse primer Rw: 5'-GAAGGCCGAGTAACCGACTT-3' (SEQ ID NO.7).

[0076] The results showed that the primers provided in this comparative example alone could not detect the SNP molecular markers in this embodiment, and the detection primers provided in this embodiment alone could not detect the SNP molecular markers provided in this comparative example. However, PCR reaction using the primer set provided by this invention could quickly detect the SNP molecular markers in the embodiment, demonstrating specificity.

[0077] Meanwhile, the mean range of the dominant and disadvantaged genotypes in body length / weight selected in this embodiment is greater than that of the previous embodiment, indicating that the embodiments of this application can accurately identify larger Litopenaeus vannamei populations, not limited to small populations, and the identification is more accurate, which can be used for subsequent Litopenaeus vannamei breeding.

[0078] Therefore, the SNP molecular marker Chr25_11409188 (SEQ ID NO.1) provided by this invention can be used to assist in the selection of fast-growing Litopenaeus vannamei broodstock. By using the AA or AG genotype in this SNP molecular marker to select broodstock, the growth rate of body length and weight 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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. The application of a reagent for detecting SNP molecular markers in the breeding of Litopenaeus vannamei, characterized in that, The SNP molecular marker is shown in SEQ ID NO.1, and the polymorphism of the 497th base from the 5' end of SEQ ID NO.1 is A / G; The breeding refers to the breeding of Litopenaeus vannamei individuals of long body length and heavy body weight, wherein the body length and weight of Litopenaeus vannamei individuals with the SNP molecular marker genotype AA are greater than the body length and weight of Litopenaeus vannamei individuals with the SNP molecular marker genotype AG, and the body length and weight of Litopenaeus vannamei individuals with the SNP molecular marker genotype GG.

2. The application of the reagent for detecting SNP molecular markers according to claim 1 in the breeding of Litopenaeus vannamei, characterized in that, The reagent is a primer pair, and the nucleotide sequence of the primer pair is as follows: Forward primer F: 5'-GTCGAGAAGAGCACCGGTAA-3'; Reverse primer R: 5'-GCAAGCCTGTTACTTCGCAA-3'.

3. The application of a kit for detecting SNP molecular markers in the breeding of Litopenaeus vannamei, characterized in that, The kit includes primer pairs, the nucleotide sequences of which are as follows: Forward primer F: 5'-GTCGAGAAGAGCACCGGTAA-3'; Reverse primer R: 5'-GCAAGCCTGTTACTTCGCAA-3'; The SNP molecular marker is shown in SEQ ID NO.1, and the polymorphism of the 497th base from the 5' end of SEQ ID NO.1 is A / G; The breeding refers to the breeding of Litopenaeus vannamei individuals of long body length and heavy body weight, wherein the body length and weight of Litopenaeus vannamei individuals with the SNP molecular marker genotype AA are greater than the body length and weight of Litopenaeus vannamei individuals with the SNP molecular marker genotype AG, and the body length and weight of Litopenaeus vannamei individuals with the SNP molecular marker genotype GG.

4. A method for breeding Litopenaeus vannamei, characterized in that, Includes the following steps: Step S1: Detect the SNP molecular markers of the Litopenaeus vannamei to obtain the genotypes of the SNP molecular markers; the SNP molecular markers are shown in SEQ ID NO.1, and the polymorphism of the 497th base from the 5' end of SEQ ID NO.1 is A / G; Step S2: Screening is performed based on the genotype of the SNP molecular marker, wherein the genotype of the SNP molecular marker is AA, AG, or GG; the screening is based on the body length and weight of the Litopenaeus vannamei. Step S3: Breeding Litopenaeus vannamei with the AA genotype using the SNP molecular marker, wherein the breeding is the breeding of long-bodied and heavy-bodied individuals of Litopenaeus vannamei.

5. The method for breeding Litopenaeus vannamei according to claim 4, 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 is performed using primer pairs to obtain PCR amplification products; Steps S1-3: Sequencing the PCR amplification products to obtain the genotype of the SNP molecular marker.

6. The method for breeding Litopenaeus vannamei according to claim 5, characterized in that, In steps S1-2, the nucleotide sequences of the primer pair are as follows: Forward primer F: 5'-GTCGAGAAGAGCACCGGTAA-3'; Reverse primer R: 5'-GCAAGCCTGTTACTTCGCAA-3'.

7. The method for breeding Litopenaeus vannamei according to claim 6, characterized in that, 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 2 min, denaturation at 98℃ for 10 s, annealing at 59℃ for 30 s, extension at 68℃ for 1 s, for a total of 30 cycles from denaturation to extension, followed by a full extension at 68℃ for 5 min.

8. The method for breeding Litopenaeus vannamei according to claim 7, characterized in that, In step S2 The body length and weight of Litopenaeus vannamei individuals with the SNP molecular marker genotype AA > the body length and weight of Litopenaeus vannamei individuals with the SNP molecular marker genotype AG > the body length and weight of Litopenaeus vannamei individuals with the SNP molecular marker genotype GG.

9. The method for breeding Litopenaeus vannamei according to claim 8, characterized in that, Step S3 specifically involves: retaining Litopenaeus vannamei with the AA genotype of the SNP molecular marker, and breeding by pairing male and female Litopenaeus vannamei with the AA genotype.

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