Application of SNP molecular marker related to nitrite tolerance trait of Oatp74D gene of penaeus vannamei
Patent Information
- Application Number
- CN202611274768.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-08-21
- Publication Date
- 2026-09-29
AI Technical Summary
但是，目前针对南美白对虾Oatp74D基因的研究多集中于序列特征与胁迫表达模式分析，尚缺乏可直接应用于育种实践的功能SNP关联位点，未见有关南美白对虾Oatp74D基因多态性与耐亚硝氮性状关联的系统研究报道
分子标记辅助育种是水产精准育种的核心技术方向，相较于传统选育，可摆脱养殖环境干扰，在生长阶段实现目标性状快速预判，有效缩短育种周期。当前南美白对虾育种已开发抗病、耐盐、耐低温等性状相关分子标记，但针对亚硝氮耐受性状的实用分子标记资源仍然稀缺。本发明通过研究Oatp74D基因上与耐亚硝氮性状显著关联的分子标记，筛选得到具有准确性高、稳定性好，筛选不受个体月龄、性别限制等优点的的SNP分子标记，再配合高效基因型检测方法，直接应用于耐亚硝氮优良亲本的快速筛选，为南美白对虾抗逆新品种培育提供有效分子工具。而且应用本发明方法无需开展大规模亚硝氮胁迫试验，即可在早期精准筛选出携带有利基因型的耐亚硝氮个体，并作为后备亲本逐级培育，大幅提高选育效率与精准度，为南美白对虾耐亚硝氮品种选育研究奠定可靠基础，有助于推动南美白对虾耐亚硝氮抗逆养殖的研究进程。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of breeding technology for Litopenaeus vannamei, specifically involving the application of a molecular marker for SNPs related to the nitrite tolerance trait of the Oatp74D gene in Litopenaeus vannamei. Background Technology
[0002] South American white shrimp ( Litopenaeus vannamei Litopenaeus vannamei (scientific name: Litopenaeus vannamei) is one of the main species in my country's marine aquaculture industry. With the rapid promotion of factory-style, high-density recirculating aquaculture systems, the continuous accumulation of uneaten feed and metabolic waste easily leads to nitrogen imbalance in the water, with nitrite becoming a frequent toxic stress factor in aquaculture systems. Nitrite can damage the respiratory function of shrimp gill tissue, interfere with the oxygen-carrying capacity of hemolymph, induce oxidative stress and hepatopancreatic damage, reduce the body's immunity, and significantly increase the risk of disease outbreaks. At present, the industry mainly relies on water quality control methods to alleviate nitrite toxicity, but the treatment cost is high and the stability is insufficient. Therefore, breeding nitrite-tolerant varieties from the germplasm level is the fundamental way to solve the industry's pain points.
[0003] Organic anion transporting polypeptide 74D (Oatp74D) belongs to the membrane transporter family, specifically the solute carrier organic anion transporter family (Oatp / Slco), and mediates sodium-independent transmembrane transport of various endogenous and exogenous organic anions. The protein encoded by this gene possesses sodium-independent organic anion transmembrane transport activity and participates in physiological processes such as ecdysone receptor signaling pathway activation and larval development. Existing research indicates that Oatp / Slco family genes play important regulatory roles in the environmental stress response of aquatic animals, mediating the transport and removal of toxic substances in water and maintaining intracellular homeostasis. However, current research on the Oatp74D gene in Litopenaeus vannamei focuses primarily on sequence characteristics and stress expression patterns, lacking functional SNP-related loci that can be directly applied to breeding practices. No systematic studies have been reported on the association between Oatp74D gene polymorphism and nitrite tolerance in Litopenaeus vannamei. Summary of the Invention
[0004] To address the aforementioned shortcomings, this invention discloses the application of SNP molecular markers related to nitrite tolerance in the Oatp74D gene of Litopenaeus vannamei. Based on screening the Oatp74D gene of Litopenaeus vannamei, SNP molecular markers related to nitrite tolerance are obtained and used as functional markers for nitrite tolerance in Litopenaeus vannamei, applied to the breeding of new nitrite-tolerant varieties of Litopenaeus vannamei.
[0005] This invention is achieved using the following technical solution: An application of a molecular marker for the Oatp74D gene of Litopenaeus vannamei related to nitrite tolerance traits: This involves detecting the genotype of the molecular marker for the SNP related to nitrite tolerance traits in Litopenaeus vannamei, and selecting individuals with the dominant genotype as reserve parents for breeding Litopenaeus vannamei nitrite-tolerant varieties. The nucleotide sequence of the SNP molecular marker related to the nitrite tolerance trait is shown in Sequence 1 of the sequence listing, wherein the 161bp site is denoted as D.351774, the base of this site is G or A, and the mutation type is G / G homozygous, A / G heterozygous, or A / A homozygous. The 166bp site is designated as D.351779. The base at this site is either T or G, and the mutation type is T / T homozygous, G / T heterozygous, or G / G homozygous. The 402bp site is designated as D.352015. The base at this site is either G or A, and the mutation type is G / G homozygous, A / G heterozygous, or A / A homozygous. The 425bp site is designated as D.352038. The base at this site is either A or T, and the mutation type is A / A homozygous, A / T heterozygous, or T / T homozygous. The 468bp site is designated as D.352081. The base at this site is either G or A, and the mutation type is G / G homozygous, A / G heterozygous, or A / A homozygous.
[0006] Furthermore, among the SNP molecular markers related to nitrite tolerance, when the genotype at the 161bp locus is AA, the individual is selected as a backup parent for breeding Litopenaeus vannamei; when the genotype at the 166bp locus is GG, the individual is selected as a backup parent for breeding Litopenaeus vannamei; when the genotype at the 402bp locus is AA or AG, the individual is selected as a backup parent for breeding Litopenaeus vannamei; when the genotype at the 425bp locus is AA or AT, the individual is selected as a backup parent for breeding Litopenaeus vannamei; and when the genotype at the 468bp locus is AA or AG, the individual is selected as a backup parent for breeding Litopenaeus vannamei.
[0007] Furthermore, genomic DNA was extracted from the muscle tissue of the Litopenaeus vannamei to be tested, and then used as template DNA for PCR amplification and purification of the PCR amplification product. The obtained product was then sequenced to determine the genotypes at the 161bp, 166bp, 402bp, 425bp, and 468bp sites on the SNP molecular markers related to the nitrite tolerance trait.
[0008] Furthermore, during the PCR amplification process, the primer combination used to detect the SNP molecular markers related to nitrite tolerance in the Oatp74D gene of Litopenaeus vannamei includes the following primers: Primer F: GTCACCCTTCGCTGTGTAGA (as shown in sequence 2 in the sequence listing); Primer R: TACCTACGCGAAGTCGGTCT (as shown in sequence 3 in the sequence listing).
[0009] Furthermore, the PCR amplification system consists of the following components: 25 μL of 2×Es TaqMasteMix, 1 μL of template DNA at a concentration of 50 ng / μL, 2 μL of primer F at a concentration of 10 μmol / L, 2 μL of primer R at a concentration of 10 μmol / L, and 20 μL of ddH2O.
[0010] Furthermore, the PCR amplification reaction procedure includes the following steps: S1. Pre-denaturate at 94℃ for 2 min; S2. Perform 35 cycles of denaturation at 94℃ for 30s, annealing at 64℃ for 30s, and extension at 72℃ for 30s. S3. Extend at 72℃ for 10 minutes.
[0011] Compared with existing technologies, this technical solution has the following advantages: Molecular marker-assisted breeding is a core technology in precision aquaculture. Compared to traditional breeding methods, it can overcome the interference of the aquaculture environment, enabling rapid prediction of target traits during the growth stage and effectively shortening the breeding cycle. Currently, molecular markers related to traits such as disease resistance, salt tolerance, and low-temperature tolerance have been developed in Litopenaeus vannamei breeding, but practical molecular marker resources for nitrite tolerance remain scarce. This invention studies molecular markers on the Oatp74D gene that are significantly associated with nitrite tolerance, screening for SNP molecular markers with high accuracy, good stability, and no limitations on individual age or sex. These markers, combined with efficient genotyping methods, can be directly applied to the rapid screening of superior nitrite-tolerant parents, providing an effective molecular tool for cultivating new stress-resistant Litopenaeus vannamei varieties. Moreover, the method of this invention does not require large-scale nitrite stress experiments. It can accurately screen individuals carrying favorable genotypes that are nitrite tolerant at an early stage and cultivate them as backup parents, which greatly improves the efficiency and accuracy of breeding. This lays a reliable foundation for the breeding research of nitrite tolerant varieties of Litopenaeus vannamei and helps to promote the research process of nitrite tolerant and stress-resistant aquaculture of Litopenaeus vannamei. Attached Figure Description
[0012] Figure 1This is a partial sequence (positions 158-164) of the product obtained from amplifying the Oatp74D gene in Example 1, showing the peak values of GG, AG, and AA at position D.351774.
[0013] Figure 2 This is a partial sequence (positions 163-169) of the product obtained from amplifying the Oatp74D gene in Example 1, showing the TT, GT, and GG peaks at position D.351779.
[0014] Figure 3 This is a partial sequence (positions 399-405) of the product obtained from amplifying the Oatp74D gene in Example 1, showing the peak values of GG, GA, and AA at position D.352015.
[0015] Figure 4 This is a partial sequence (positions 422-428) of the product obtained from amplifying the Oatp74D gene in Example 1, showing the peak values of AA, AT, and TT at position D.352038.
[0016] Figure 5 This is a partial sequence (positions 465-471) of the product obtained from amplifying the Oatp74D gene in Example 1, showing the peak values of GG, AG, and AA at position D.352081. Detailed Implementation
[0017] The present invention is further illustrated by the following examples, but these are not intended to limit the invention. Specific experimental conditions and methods not specified in the following examples are generally conventional methods well known to those skilled in the art.
[0018] Example 1: The screening process for SNP molecular markers related to nitrite tolerance in the Oatp74D gene of Litopenaeus vannamei described in this invention is as follows: (1) Several plastic cylindrical containers with a volume of 1000 L were used. 500 L of culture water was poured into each container, and analytical grade sodium nitrite was added. The container was stirred thoroughly to ensure complete dissolution, and the final concentration of sodium nitrite in the water was adjusted to 757.18 mg / L. In addition, plastic cylindrical containers of the same volume were used as negative controls, i.e., only 500 L of culture water was poured into each container, but no sodium nitrite was added. Other culture conditions were the same. 240 whiteleg shrimp weighing about 10 g were selected and temporarily raised for 3 days. They were then randomly distributed into each plastic cylindrical container at a density of 30 shrimp per container to conduct an acute nitrite stress experiment. During the experiment, the culture water was continuously aerated, and the water temperature was controlled at 27±0.5℃, pH at 8.2±0.3, salinity at 30.0‰, and dissolved oxygen at 7~8 mg / L. To maintain the stress concentration, all water was replaced every 24 hours and sodium nitrite was added again to 757.18 mg / L. mg / L, negative control only changed the water without adding sodium nitrite; feeding was stopped throughout the experiment to maintain the experimental nitrite concentration; after the shrimp rolled over, they were touched with a stick, and if they could not swim away quickly or showed no obvious escape reaction and continued to maintain the rolled-over posture, they were considered dead; mortality was recorded starting 3 hours after the stress began, and the earliest dead individuals (45) within 3-12 hours after stress and the individuals still alive and in the best condition after 96 hours of stress (45) were collected as experimental samples for the nitrite-sensitive group and the nitrite-tolerant group; the negative control showed no dead individuals, indicating that sodium nitrite was the cause of death in shrimp in the acute stress experiment.
[0019] (2) Extract total DNA from the muscle tissue of Litopenaeus vannamei from all samples obtained in step (1) using the ammonium acetate / isopropanol method. Detect the quality and integrity of the extracted DNA using an ultra-micro UV spectrophotometer and agarose gel electrophoresis. Store the obtained total DNA at -20°C for later use. In daily breeding and farming, DNA extraction and genotype detection can be performed using the appendage muscle tissue of Litopenaeus vannamei, which has minimal impact on the health of the shrimp.
[0020] (3) Primer combinations were designed based on the Oatp74D gene sequence of Litopenaeus vannamei, and then SNP sites located in the Oatp74D gene were amplified and screened. The primer combination includes the following primers: The sequence of primer F is: GTCACCCTTCGCTGTGTAGA; The sequence of primer R is: TACCTACGCGAAGTCGGTCT; The PCR amplification system consists of the following components: 25 μL of 2×Es Taq MasteMix, 1 μL of template DNA at a concentration of 50 ng / μL, 2 μL of primer F at a concentration of 10 μmol / L, 2 μL of primer R at a concentration of 10 μmol / L, and 20 μL of ddH2O. The PCR amplification reaction procedure includes the following steps: S1. Pre-denaturate at 94℃ for 2 min; S2. Perform 35 cycles of denaturation at 94℃ for 30s, annealing at 64℃ for 30s, and extension at 72℃ for 30s. S3. Extend at 72℃ for 10 minutes.
[0021] (4) The PCR amplification products were purified and sequenced after detection by 1% agarose gel electrophoresis. The sequencing results were compared and analyzed using DNAstar software, including nucleotide sequence alignment and peak analysis (e.g., Figures 1-5 As shown in the figure, relevant SNP sites were screened out; the PCR amplification product sequence of one sample is shown below: ; The 161 bp site is designated as D.351774, the 166 bp site as D.351779, the 402 bp site as D.352015, the 425 bp site as D.352038, and the 468 bp site as D.352081.
[0022] (5) Based on the selected SNP sites, the Litopenaeus vannamei in the sensitive group and the tolerant group were detected and genotyped according to the above method. The samples of different SNP sites in the sensitive group and the tolerant group were counted, the genotype frequency and allele frequency were calculated, and the independence test was performed by chi-square analysis. The specific results are shown in Table 1.
[0023] Analysis of Table 1 shows that genotypic polymorphism at the marked loci has a significant impact on the nitrite tolerance trait in Litopenaeus vannamei, as detailed below: Individuals with the AA genotype at the 161bp locus are individuals with a dominant nitrite tolerance trait and can be selected as backup parents for breeding of Litopenaeus vannamei varieties. Individuals with the GG genotype at the 166bp locus are individuals with a dominant nitrite tolerance trait and can be selected as backup parents for breeding of Litopenaeus vannamei varieties. Individuals with the AA or AG genotype at the 402bp locus are individuals with a dominant nitrite tolerance trait and can be selected as backup parents for breeding of Litopenaeus vannamei varieties. Individuals with the AA or AT genotype at the 425bp locus are individuals with a dominant nitrite tolerance trait and can be selected as backup parents for breeding of Litopenaeus vannamei varieties. Individuals with the AA or AG genotype at the 468bp locus are individuals with a dominant nitrite tolerance trait and can be selected as backup parents for breeding Litopenaeus vannamei varieties.
[0024] Table 1. Genotype and allele frequency distribution at different loci in the target fragment of the Oatp74D gene ( P <0.05 indicates a significant correlation.
[0025] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.
Claims
1. The application of a molecular marker for the nitrite tolerance trait associated with the Oatp74D gene in Litopenaeus vannamei, characterized in that: The application involves detecting the genotypes of SNP molecular markers related to nitrite tolerance in Litopenaeus vannamei, and selecting individuals with dominant genotypes as reserve parents for breeding Litopenaeus vannamei nitrite-tolerant varieties. The nucleotide sequences of the SNP molecular markers related to nitrite tolerance are shown in Sequence 1 of the sequence listing. The 161 bp site is designated as D.351774, with a base of G or A, and the mutation types are G / G homozygous, A / G heterozygous, and A / A homozygous. The 166 bp site is designated as D.351779, with a base of T or G, and the mutation types are T / T homozygous, G / T heterozygous, and G / G homozygous. The 402bp site is designated D.352015, with a base of G or A, and the mutation types are G / G homozygous, A / G heterozygous, and A / A homozygous; the 425bp site is designated D.352038, with a base of A or T, and the mutation types are A / A homozygous, A / T heterozygous, and T / T homozygous; the 468bp site is designated D.352081, with a base of G or A, and the mutation types are G / G homozygous, A / G heterozygous, and A / A homozygous.
2. The application according to claim 1, characterized in that: Among the SNP molecular markers related to nitrite tolerance, when the genotype at the 161bp locus is AA, the individual is selected as a backup parent for breeding Litopenaeus vannamei; when the genotype at the 166bp locus is GG, the individual is selected as a backup parent for breeding Litopenaeus vannamei; when the genotype at the 402bp locus is AA or AG, the individual is selected as a backup parent for breeding Litopenaeus vannamei; when the genotype at the 425bp locus is AA or AT, the individual is selected as a backup parent for breeding Litopenaeus vannamei; and when the genotype at the 468bp locus is AA or AG, the individual is selected as a backup parent for breeding Litopenaeus vannamei.
3. The application according to claim 1, characterized in that: Genomic DNA was extracted from the muscle tissue of the Litopenaeus vannamei to be tested, and then used as template DNA for PCR amplification and purification of the PCR amplification products. The obtained products were then sequenced to determine the genotypes at the 161bp, 166bp, 402bp, 425bp, and 468bp sites on the SNP molecular markers related to the nitrite tolerance trait.
4. The application according to claim 3, characterized in that: During the PCR amplification process, the primer combination used to detect the SNP molecular markers related to nitrite tolerance in the Oatp74D gene of Litopenaeus vannamei includes the following primers: Primer F: GTCACCCTTCGCTGTGTAGA; Primer R: TACCTACGCGAAGTCGGTCT.
5. The application according to claim 4, characterized in that: The PCR amplification system consists of the following components: 25 μL of 2×Es Taq MasteMix, 1 μL of template DNA at a concentration of 50 ng / μL, 2 μL of primer F at a concentration of 10 μmol / L, 2 μL of primer R at a concentration of 10 μmol / L, and 20 μL of ddH2O.
6. The application according to claim 3, characterized in that: The PCR amplification reaction procedure includes the following steps: S1. Pre-denaturate at 94℃ for 2 min; S2. Perform 35 cycles of denaturation at 94℃ for 30s, annealing at 64℃ for 30s, and extension at 72℃ for 30s. S3. Extend at 72℃ for 10 minutes.