A marker and method for screening fast-growing individuals of epinephelus fuscoguttatus and hybrid offspring in high-density culture and application thereof

Abstract

The application belongs to the technical field of aquatic animal molecular biology, and particularly relates to a marker and a method for screening fast-growing individuals of Epinephelus fuscoguttatus and hybrid offspring under high-density culture and application. The marker sites of the fast-growing individuals of Epinephelus fuscoguttatus and hybrid offspring are SNP1 (chr11_14941311), SNP2 (chr9_45571123), SNP3 (chr4_34158127), InDel1 (chr1_28273434) and InDel2 (chr5_19085475). By using the joint genotype assisted determination method of the application, individuals or parents with fast growth potential under high-density culture conditions can be quickly identified in the early stage of Epinephelus fuscoguttatus and Epinephelus lanceolatus breeding, excellent individual selection is realized in the stage of grouper seed cultivation, excellent groups tolerating high-density culture are screened, the cost of culture is reduced, and the efficiency of industrialized culture is improved.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology of aquatic animals, and specifically relates to a marker, method and application for screening brown-spotted grouper and fast-growing individuals of hybrid offspring in high-density aquaculture. Background Technology

[0002] Brown-spotted grouper ( Brown-marbled grouper It is one of my country's important grouper germplasm resources and plays a vital role in the genetic improvement and hybridization breeding of grouper. It is compared with the saddle-banded grouper (also known as the giant grouper). Giant grouper The offspring of the pearl grouper obtained by hybridization ( Epinephelus fuscoguttatus ♀× Giant grouper ♂ (male) hybrid offspring possess characteristics such as rapid growth, strong disease resistance, and good environmental adaptability, making them a promising candidate for the market and a key target for industrialized recirculating aquaculture systems. However, with the continuous expansion of high-density intensive aquaculture, while increasing yields, a series of negative reactions have also been induced. Fish typically face continuous stress from stronger feeding competition and limited space, leading to problems such as increased individual growth differentiation and decreased growth performance. Even within the same aquaculture population, the adaptability of different individuals under long-term high-density aquaculture stress often varies significantly. Some individuals exhibit stronger adaptability to the stressful environment and can maintain a relatively fast growth rate, while others show stunted growth or even death under stress. Therefore, in the specific context of high-density aquaculture, identifying parents or individuals with good growth potential in the early stages of breeding is crucial for improving aquaculture efficiency and cultivating superior strains suitable for high-density aquaculture.

[0003] Current strategies for addressing high-density stress primarily rely on aquaculture management and control, such as optimizing stocking density, improving water quality, or adding functional nutrient additives. While these measures can alleviate stress symptoms to some extent, they are passive responses and cannot fundamentally improve the breed's own tolerance. Traditional selective breeding relies on phenotypic data and family selection. Selecting for the complex quantitative trait of high-density tolerance has inherent limitations, including long cycles, high costs, and low accuracy. Furthermore, it is easily affected by environmental factors, and traditional methods struggle to accurately fix dominant genotypes.

[0004] With the development of molecular biology and high-throughput sequencing technologies, genome-wide association studies (GWAS) have been successfully applied to the screening of molecular markers for growth, disease resistance, and some stress resistance traits in cultured organisms, validating the feasibility of this technical approach. Single nucleotide polymorphisms (SNPs) or insertion-deletion sites (InDels) are widely used in trait association analysis, population genetic analysis, and marker-assisted selection due to their rich polymorphism, stable detection, and suitability for large-scale analysis. Utilizing molecular markers for early screening of target traits and assisted breeding has become an important technical route for improving the breeding efficiency of aquatic animals. Biologically, the pearl grouper, as a hybrid offspring of female brown-spotted grouper and male grouper, inherits the nuclear genome information of the brown-spotted grouper. Therefore, the two have a clear correspondence in corresponding homologous chromosomal regions and genetic loci, which provides a biological basis for the detection, typing, and comparative analysis of relevant loci in brown-spotted grouper and its hybrid offspring. In existing technologies, some studies and patents have disclosed molecular markers related to disease resistance and stress tolerance traits, and have attempted to use them for early auxiliary screening of economic traits.

[0005] However, existing technologies still have shortcomings: on the one hand, existing research has mostly focused on the development of molecular markers related to certain disease resistance or stress resistance traits, and still lacks molecular marker-assisted screening schemes for the specific application scenario of rapid growth traits in grouper under high-density aquaculture conditions. On the other hand, existing technologies mostly use single-type molecular markers as auxiliary screening criteria. However, if only SNP sites are used, although they are abundant, they can only reflect single nucleotide substitution information; if only InDel sites are used, although they can be used for relatively intuitive typing through fragment length differences, they cannot fully reflect the genetic information at the level of single base substitution. In high-density aquaculture scenarios, rapid growth is a complex trait influenced by multiple genes and environmental factors. Relying solely on single-type molecular markers for auxiliary screening often results in insufficient coverage of genetic information, limited screening dimensions, and insufficient differentiation of candidate individuals. Therefore, it is necessary to develop a joint screening method for high-density grouper aquaculture scenarios to conduct early auxiliary assessment and screening of the rapid growth potential of test individuals under high-density aquaculture conditions. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a marker, method, and application for screening brown-spotted grouper and its rapidly growing hybrid offspring in high-density aquaculture.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A marker for screening brown-spotted grouper and its hybrid offspring for high-density aquaculture is proposed. The marker loci for brown-spotted grouper and its hybrid offspring for rapid growth are SNP1 (chr11_14941311), SNP2 (chr9_45571123), SNP3 (chr4_34158127), InDel1 (chr1_28273434), and InDel2 (chr5_19085475).

[0008] A method for screening brown-spotted grouper and fast-growing hybrid offspring using the aforementioned markers in high-density aquaculture. (1) Cut off the tail fin rays of the brown spot grouper or its hybrid offspring (pearl giant grouper) under high-density culture and extract their individual genomic DNA; (2) Detect the genotype of the individual to be tested at the following core loci: SNP1 (chr11_14941311), SNP2 (chr9_45571123), SNP3 (chr4_34158127), InDel1 (chr1_28273434) and InDel2 (chr5_19085475); (3) Based on the genotype of the above 5 core loci, assist in identifying whether the tested individual is a candidate individual with rapid growth potential under high-density breeding conditions.

[0009] The hybrid offspring were brown-spotted grouper ( Brown-marbled grouper ) and the saddle-banded grouper (also known as the giant grouper, Giant grouper The offspring of the pearl grouper obtained through hybridization.

[0010] The specific high-density aquaculture scenario requires a stocking density of 40-50 kg / m². 3 .

[0011] When the core locus of the individual to be tested is the following dominant genotype, it can be identified as a candidate for rapid growth under high-density breeding conditions: SNP1 is AG or GG type; SNP2 is AG or GG type; SNP3 is AG or AA type; InDel1 is ID or II type; InDel2 is ID or II type.

[0012] The above SNP sites are A / G polymorphic sites, and their possible genotypes include AA, AG and GG. The InDel site is an insertion site, where I represents an insertion allele and D represents a non-insertion allele.

[0013] The InDel1 site represents the insertion of the base TTA; the InDel2 site represents the insertion of the base A.

[0014] The primers used in step (2) to detect SNP1, SNP2, SNP3, InDel1, and InDel2 sites are as follows: SNP1_F: TGGCTTCACTTGACCAAAAAAGA; SNP1_R: AATTATTGGACCTGCACCCGG; SNP2_F: GCCGCCATGTAAAGTGTGTG; SNP2_R:CACTGTGTTACGCTTGACTATTCAT; SNP3_F:TTGAGAGCATCATAAAGAAAGACAGTT; SNP3_R: ATAATAATAATAATAATAATAATTTAAAAAAAAGTTATCTT; InDel1_F:ACACAAGAAGACAAAATAAGTTTGTATTATTT; InDel1_R:AGGAGTTATTGTAGTATTTTTAAAATTGATTT; InDel2_F:AGGGTAGAGAGGAGAGCGAGA; InDel2_R: TGGAACTGTTTTAAATTGGGTGC.

[0015] Using the primers, PCR amplification was performed, and genotyping was performed at 5 core loci. When the tested individual exhibited a dominant genotype combination at the 5 core loci, it could be identified as a candidate individual of grouper that grows rapidly under high-density aquaculture conditions.

[0016] One application of the marker is its use in high-density aquaculture conditions for assisted screening of brown-spotted grouper and rapidly growing candidate individuals of hybrid offspring, assisted selection of parents, or molecular marker-assisted breeding.

[0017] One application of the method is the selection of individuals for use as fast-growing candidate individuals in assisted breeding.

[0018] Advantages of this invention This invention screens SNP and InDel loci that are significantly associated with rapid growth traits under high-density aquaculture conditions and establishes corresponding joint typing-assisted screening rules. This helps improve the efficiency and stability of early auxiliary assessment and screening of candidate individuals, enabling auxiliary identification and screening of candidate individuals in the early stages of breeding. This improves the screening efficiency of rapid-growing candidate individuals under high-density aquaculture conditions and provides technical support for molecular marker-assisted breeding of brown-spotted grouper and its hybrid offspring. Attached Figure Description

[0019] Figure 1 Manhattan plot for genome-wide association analysis of pearl grouper. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are described in detail below with reference to specific embodiments. However, the scope of this invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to this invention without departing from the spirit and scope thereof.

[0021] Example 1: Screening of SNPs and InDel molecular markers related to rapid growth traits in brown-spotted grouper and its hybrid offspring (pearl grouper) in high-density aquaculture scenarios based on genome-wide association analysis. 1. Sample source and experimental treatment of pearl grouper (1) The pearl grouper juveniles (48.6±8.2g, 14.4±1.5cm) used in the experiment were all purchased from Shenzhen Haijuyuan Aquatic Technology Co., Ltd. Grouper of uniform size and in good health were introduced at 40kg / m 3 The fish were subjected to stocking density stress for 60 days, and the weight of each fish was recorded at the last moment.

[0022] (2) The weight data were distributed normally to distinguish between fast-growing and slow-growing individuals. The top 25 fast-growing individuals and the bottom 25 slow-growing individuals were selected, and their caudal fin rays were cut and flash-frozen in liquid nitrogen and stored in an ultra-low temperature freezer for genomic DNA extraction.

[0023] (3) Whole genome sequencing of pearl grouper was completed at Hangzhou Lianchuan Biotechnology Co., Ltd. After obtaining the initial data, genome-wide association analysis of fast / slow growth traits was performed. Figure 1 ).

[0024] 2. Experimental Results (1) The five most significant associated sites with the highest -log10(P) values ​​were obtained, which are the SNP / InDel sites associated with the rapid growth traits of brown spot grouper and pearl giant grouper in high-density aquaculture scenarios.

[0025] (2) Information on the 5 core sites: SNP1 is located at position 14941311 on chromosome 11 and is an A / G allele mutation; SNP2 is located at position 45571123 on chromosome 9 and is an A / G allele mutation; SNP3 is located at position 34158127 on chromosome 4 and is an A / G allele mutation; The InDel1 site is located at position 28273434 on chromosome 1 and is an insertion of the base TTA. The InDel2 site is located at position 19085475 on chromosome 5 and is an insertion of base A.

[0026] Example 2: PCR validation analysis of SNP / InDel loci associated with rapid growth traits in brown-spotted grouper and pearl grouper in high-density aquaculture settings. 1. Experimental Methods (1) Extract the base sequence of 5 core sites Based on the location information of the five core sites obtained in Example 1, the upstream and downstream sequences of the five core sites in the genome of the brown spot grouper (mother) which is most closely related to the pearl grouper were retrieved from the database. The sequence information is shown in Table 1.

[0027] Table 1: Information on 5 core loci significantly associated with rapid growth traits in brown-spotted grouper and pearl grouper.

[0028] (2) Design primers Based on the above sequence information, site primers were designed. The primer information is as follows: SNP1_F: TGGCTTCACTTGACCAAAAAAGA (Sequence 6); SNP1_R: AATATTGGACCTGCACCCGG (Sequence 7); SNP2_F: GCCGCCATGTAAAGTGTGTG (Sequence 8); SNP2_R: CACTGTGTTACGCTTGACTATTCAT (Sequence 9); SNP3_F: TTGAGAGCATCATAAAGAAAGACAGTT (Sequence 10); SNP3_R: ATAATAATAATAATAATAATAATTTAAAAAAAAGTTATCTT (sequence 11); InDel1_F: ACACAAGAAGACAAAATAAGTTTGTATTATTT (Sequence 12); InDel1_R: AGGAGTTATTGTAGTATTTTTAAAATTGATTT (sequence 13); InDel2_F: AGGGTAGAGAGAGAGGAGAGCGAGA (Sequence 14); InDel2_R: TGGAACTGTTTTAAATTGGGTGC (Sequence 15).

[0029] (3) PCR amplification The molecular marker amplification system described above consisted of: 2 μL DNA template (50 ng / μL), 12.5 μL Premix Tag enzyme, 1 μL each of forward and reverse primers, and ddH2O to a final volume of 25 μL. The PCR reaction conditions were: 98℃ pre-denaturation for 3 min, 35 amplification cycles (98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 10 s), and a final extension at 72℃ for 5 min.

[0030] (4) Genotyping The PCR products were sequenced, and the genotypes of five core loci were obtained based on the sequencing results. The association between the genotyping results and the rapid growth traits of the brown-spotted grouper and the pearl grouper was analyzed.

[0031] 2. Experimental Results The differences in alleles and genotypes at the five core loci between the two groups are shown in Table 2.

[0032] Table 2: Genotype and allele frequencies of the five core loci between the two groups

[0033] In summary, the combined genotypes described in this invention include five SNP / InDel molecular marker loci and their genotype combinations on chromosomes 11, 9, 4, 1, and 5 of the brown-spotted grouper and pearl grouper. Using the combined genotype-assisted determination method of this invention, individuals or parents with rapid growth potential under high-density aquaculture conditions can be quickly identified in the early stages of brown-spotted grouper and pearl grouper breeding. This allows for the selection of superior individuals during the grouper fry rearing stage, assists in screening superior populations tolerant of high-density aquaculture, reduces aquaculture costs, and improves the efficiency of factory farming of brown-spotted grouper and pearl grouper.

[0034] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A marker for screening brown-spotted grouper and its hybrid offspring for high-density aquaculture, characterized in that: The marker loci for the brown-spotted grouper and its hybrid offspring that grow rapidly are SNP1 (chr11_14941311), SNP2 (chr9_45571123), SNP3 (chr4_34158127), InDel1 (chr1_28273434), and InDel2 (chr5_19085475).

2. A method for screening brown-spotted grouper and rapidly growing hybrid offspring using the marker described in claim 1 for high-density aquaculture, characterized in that: (1) Cut off the tail fin rays of the brown spot grouper or its hybrid offspring under high-density culture and extract their individual genomic DNA; (2) Detect the genotype of the individual to be tested at the following core loci: SNP1 (chr11_ 14941311), SNP2 (chr9_45571123), SNP3 (chr4_34158127), InDel1 (chr1_28273434) and InDel2 (chr5_19085475); (3) Based on the genotype of the above 5 core loci, assist in identifying whether the tested individual is a candidate individual with rapid growth potential under high-density breeding conditions.

3. The method according to claim 2, characterized in that: The hybrid offspring were brown-spotted grouper ( Epinephelus fuscoguttatus ) and saddle-banded grouper ( Epinephelus lanceolatus The offspring of the pearl grouper obtained through hybridization.

4. The method according to claim 2, characterized in that, The specific high-density aquaculture scenario requires a stocking density of 40-50 kg / m³. 3 .

5. The method according to claim 2, characterized in that: When the core loci of the individual to be tested are the following dominant genotypes, it can be identified as a candidate for rapid growth under high-density breeding conditions: SNP1 is AG or GG type; SNP2 is AG or GG type; SNP3 is AG or AA type; InDel1 is ID or II type; InDel2 is ID or II type.

6. The method according to claim 2, characterized in that, Includes primer combinations for detecting SNP1, SNP2, SNP3, InDel1, and InDel2 sites: SNP1_F: TGGCTTCACTTGACCAAAAAAGA; SNP1_R: AATTATTGGACCTGCACCCGG; SNP2_F: GCCGCCATGTAAAGTGTGTG; SNP2_R:CACTGTGTTACGCTTGACTATTCAT; SNP3_F:TTGAGAGCATCATAAAGAAAGACAGTT; SNP3_R: ATAATAATAATAATAATAATAATTTAAAAAAAAGTTATCTT; InDel1_F:ACACAAGAAGACAAAATAAGTTTGTATTATTT; InDel1_R:AGGAGTTATTGTAGTATTTTTAAAATTGATTT; InDel2_F:AGGGTAGAGAGGAGAGCGAGA; InDel2_R: TGGAACTGTTTTAAATTGGGTGC.

7. The method according to any one of claims 2-6, characterized in that, PCR amplification was performed using the primer combination, and genotyping was performed at 5 core loci. When the tested individual exhibited a dominant genotype combination at the 5 core loci, it could be identified as a candidate grouper individual with rapid growth traits under high-density aquaculture conditions.

8. An application of the mark according to claim 1, characterized in that: The markers are used in the auxiliary screening of rapidly growing candidate individuals of brown-spotted grouper and its hybrid offspring, the auxiliary selection of parents, or the application of molecular marker-assisted breeding under high-density aquaculture conditions.

9. An application of the method according to claim 2, characterized in that, The method screens individuals for use as fast-growing candidate individuals in assisted breeding.

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