Application of culter alburnus molecular marker combination, chip, kit, method and application

Abstract

The invention relates to the technical field of molecular markers, and particularly discloses application of a culter alburnus molecular marker combination as well as a chip, a kit, a method and application of the culter alburnus molecular marker combination, the molecular marker combination comprises 12263 SNP sites, and the physical positions of the 12263 SNP sites are determined based on whole genome sequence alignment of a culter alburnus reference genome. The version number of the whole genome sequence of the culter alburnus reference genome is GWHBOSX00000000. Fa, and the version number of the whole genome sequence of the culter alburnus reference Wherein the physical position information of the 12263 SNP sites is specifically shown in the following table 1. The molecular marker combination disclosed by the invention can be used for carrying out genetic typing on culter alburnus; the method can be used for detecting the economic characters of the culter alburnus and realizing whole genome association analysis of the culter alburnus and breeding and auxiliary breeding of the culter alburnus; economic characters of culter alburnus include weight, body length, body height, viscera weight, viscera-removed weight, full length, tail length, fatness and viscera removal rate.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, specifically to the uses, chips, kits, methods, and applications of molecular marker combinations for Culter alburnus. Background Technology

[0002] Culter alburnus ( Culter alburnus Basilewsky belongs to the subfamily Cultinae and the genus Cultinae.

[0003] Due to the high egg production of the Culter alburnus, inbreeding depression easily occurs under artificial breeding conditions. Through generations of breeding, problems such as decreased heterozygosity, poor resistance to adverse conditions, and phenotypic degeneration in farmed Culter alburnus populations have become increasingly prominent. This has led to genetic degradation phenomena in farmed Culter alburnus, including decreased growth, smaller size, and precocious sexual maturity. For a long time, breed improvement of Culter alburnus has mainly relied on traditional breeding methods such as population selection, family selection, and distant hybridization. Although these methods have achieved some success in improving growth rate and body shape, they still have significant limitations: long cycles, low efficiency, insufficient accuracy and precision, and insufficient utilization of molecular-level information. Genetic improvement has become an urgent need for the sustainable and healthy development of Culter alburnus farming.

[0004] With the theoretical research and algorithm optimization of genome selection technology, it will be applied to more fish growth, disease resistance, stress resistance and high-quality breeding, and is expected to develop into a new mainstream molecular breeding technology for fish genetic breeding.

[0005] With the rapid development of molecular biology and genomics technologies, single nucleotide polymorphisms (SNPs) have emerged as the most valuable molecular markers. SNPs are numerous, widely distributed, and genetically stable in the genomes of various species, making them ideal tools for genetic mapping, QTL (quantitative trait loci) localization, genetic diversity analysis, and population genetic structure assessment. Liquid chips, especially high-density SNP chips based on high-throughput platforms, can simultaneously, rapidly, and accurately genotype thousands of SNP loci, greatly improving the efficiency and throughput of genotyping. Applying this technology to the breeding of Culter alburnus has the following advantages: 1. Achieving precise molecular breeding: High-density chips with 10K (ten thousand) SNP loci can cover most regions of the Culter alburnus genome, providing a solid foundation for genome-wide association studies (GWAS) and fine-mapping of genes or QTLs affecting important economic traits (such as growth, disease resistance, low spine count, and feed efficiency). 2. Accelerated Breeding Process: Based on microarray genotyping data, marker-assisted selection (MAS) and genome-wide selection (GS) can be implemented. Breeders can analyze the genotypes of juvenile fish to predict their adult performance potential early and accurately, thereby significantly shortening generation intervals and accelerating the breeding cycle. 3. Efficient Germplasm Resource Management: SNP microarrays can be used to conduct detailed assessments of the genetic diversity of existing Culter alburnus populations or families, monitor inbreeding coefficients, avoid genetic degradation, and provide data support for constructing scientifically sound core populations for conservation and breeding.

[0006] Currently, although molecular genetic breeding research on the Culter alburnus has made breakthroughs in cutting-edge technologies such as gene editing (e.g., gene knockout), both domestically and internationally, significant progress has been made. mstn Genes are used to improve growth traits and for research bmp6 While the relationship between genes and intermuscular spines is well understood, there is still a gap in the development of medium-to-high density, large-scale, application-oriented SNP liquid-phase microarray tools. Existing genetic markers are often limited in number or have poor platform compatibility, making it difficult to support complex genome-wide selection breeding work. Therefore, developing a liquid-phase microarray containing 10,000 rigorously screened SNP loci that are highly associated with important economic traits in Culter alburnus or are evenly distributed throughout the genome will be a key technological tool for promoting the transformation and upgrading of Culter alburnus from traditional breeding to precision molecular breeding. Summary of the Invention

[0007] The purpose of this invention is to provide the uses, chips, kits, methods, and applications of molecular marker combinations for Culter alburnus. The molecular marker combinations of this invention can be used for genotyping of Culter alburnus; can be used to detect economic traits of Culter alburnus; and can be used to realize genome-wide association analysis of Culter alburnus, as well as breeding and assisted breeding of Culter alburnus.

[0008] This invention is achieved through the following technical solution: The uses of a molecular marker combination for Culter alburnus include applications in Culter alburnus genotyping, in detecting economic traits of Culter alburnus, in genome-wide association studies of Culter alburnus, in Culter alburnus breeding, or in Culter alburnus assisted breeding; economic traits of Culter alburnus include body weight, body length, body height, visceral weight, viscerated weight, total length, tail length, condition factor, and visceration rate; The molecular marker combination includes 12,263 SNP sites. The physical locations of these 12,263 SNP sites were determined based on whole-genome sequence alignment of the *Culter alburnus* reference genome, version number GWHBOSX00000000.fa. The specific physical location information of the 12,263 SNP sites is shown in Table 1 below. Table 1

[0009] In the physical location section, the number before the underscore represents the chromosome. ， The number after "_" indicates the location of the locus on the corresponding chromosome.

[0010] The 12,263 SNP loci of this invention are obtained by using Culter alburnus from different regions as parents, obtaining juvenile fish through natural reproduction, collecting DNA samples from the juvenile fish, and obtaining the DNA samples through whole-genome resequencing and GWAS analysis. The 12,263 SNP loci can be used to genotype Culter alburnus, detect the economic traits of Culter alburnus, and realize genome-wide association analysis, breeding, and assisted breeding of Culter alburnus.

[0011] The application of a molecular marker combination of Culter alburnus in a gene breeding chip, which includes a liquid phase chip.

[0012] A 10K liquid-phase chip for Culter alburnus, the genotyping of which includes 12,263 SNP loci.

[0013] Furthermore, the Culter alburnus 10K liquid phase chip also includes probes designed based on gene sequences covering 12,263 SNP sites.

[0014] Furthermore, the probe sequence has a GC content of 30-70%, a probe length of 110 bp, a maximum upper limit of less than 5 specific similar fragments on the reference genome, and a maximum distance of less than 10 bp from the designed region.

[0015] A kit comprising the above-mentioned Culter alburnus 10K liquid phase chip or comprising probes and / or primers for detecting 12,263 SNP sites as shown in Table 1.

[0016] A design method for a 10K liquid phase chip for Culter alburnus includes the following steps: S1. Obtaining high-quality SNP sites: S11. Sample collection: Using Culter alburnus from different regions as parents, juvenile fish are obtained through natural reproduction, and DNA samples of the juvenile fish are collected. S12. Obtaining raw SNP sites: Whole genome resequencing of DNA samples, preliminary filtering of raw reads, comparison and screening with the Culter alburnus reference genome to obtain raw SNP sites; S13. Perform quality control filtering on the original SNP sites to obtain high-quality SNP sites; S2, Functional site screening: S21. Obtain phenotypic information related to the economic traits of Culter alburnus from the juvenile fish used for DNA sample collection. S22. Perform quality control on phenotypic information; S23. GWAS analysis was performed on high-quality SNP loci and phenotypic information after quality control; SNP loci related to economic traits of Culter alburnus were obtained, and 2108 functional SNP loci were screened; the 2108 functional SNP loci include 142 loci related to body weight, 527 loci related to body length, 1020 loci related to body height, 155 loci related to visceral weight, 114 loci related to body weight without visceral, 25 loci related to total length, 18 loci related to tail length, 6 loci related to condition factor (CF), and 218 loci related to gutted yield (GY).

[0017] S3, Background Site Filtering: From the SNP sites obtained through comparison and screening of the Culter alburnus reference genome, 10,155 background SNP sites were obtained, which were combined with 2,108 functional SNP sites to form 12,263 SNP sites; S4. Probe Design: A probe sequence designed to detect 12,263 SNP sites.

[0018] Application of a 10K liquid phase chip for detecting the genotype of Culter alburnus. Genotype detection includes genotyping.

[0019] An application of a 10K liquid phase chip for detecting the economic traits of Culter alburnus, wherein the economic traits include body weight, body length, body height, visceral weight, viscerated weight, total length, tail length, fatness and visceration rate.

[0020] The application of a 10K liquid phase chip for Culter alburnus in Culter alburnus breeding, genome-wide association analysis, and assisted breeding. Culter alburnus breeding includes population selection and selection of superior varieties.

[0021] Application of the kit in detecting the genotype of Culter alburnus.

[0022] The kit is used to detect the economic traits of Culter alburnus, which include body weight, body length, body height, visceral weight, viscerated weight, total length, tail length, condition factor, and visceration rate.

[0023] The kit's applications include those in Culter alburnus breeding, genome-wide association studies, and assisted breeding. Culter alburnus breeding encompasses both population selection and the selection of superior varieties.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. This invention uses Culter alburnus from different regions as parents, obtains juvenile fish through natural reproduction, collects DNA samples from the juvenile fish, and obtains molecular marker combinations including 12,263 SNPs through whole-genome resequencing and GWAS analysis of the juvenile fish DNA samples; and develops a Culter alburnus 10K liquid phase chip and kit based on the 12,263 SNP loci. The molecular marker combinations, Culter alburnus 10K liquid phase chip and kit can be used for Culter alburnus genotyping, Culter alburnus breeding or assisted breeding, detection of Culter alburnus DNA samples, detection of economic traits of Culter alburnus, and genome-wide association analysis of Culter alburnus.

[0025] 2. The Culter alburnus 10K liquid phase chip developed in this invention is of great significance for improving the utilization efficiency of germplasm resources, accelerating the breeding process of superior traits, and enhancing the economic value and environmental adaptability of aquaculture varieties. The Culter alburnus 10K liquid phase chip of this invention adopts the latest GBTS targeted sequencing genotype detection technology for marker genotype detection, which has the advantages of low cost, high accuracy, and high detection sensitivity. Attached Figure Description

[0026] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings: Figure 1 This is a map showing the distribution of marker chromosomes for the 12,263 SNP loci provided in Embodiment 1 of the present invention; Figure 2 This is a distribution statistics chart of the 12,263 SNP sites provided in Embodiment 1 of the present invention; Figure 3 This is a gene structure distribution diagram of the 12,263 SNP sites provided in Example 1 of the present invention; Figure 4 The site detection rate of the 10K liquid phase chip for Culter alburnus provided in Embodiment 2 of the present invention; Figure 5 This is a distribution map of Culter alburnus genome breeding values ​​(body weight) obtained by whole-genome selection breeding of Culter alburnus using the 10K liquid phase chip provided in Embodiment 3 of the present invention. Figure 6 This is a distribution map of the Culter alburnus genome breeding values ​​(body length) obtained by using the 10K liquid phase chip provided in Embodiment 3 of the present invention for whole-genome selection breeding of Culter alburnus. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The illustrative embodiments and descriptions of this invention are for illustrative purposes only and are not intended to limit the invention. The embodiments described below are some, but not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0028] In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other embodiments, well-known structures, materials, or methods are not specifically described to avoid obscuring the invention. Unless otherwise specified, the materials, instruments, and reagents used in the following embodiments are commercially available. Unless otherwise specified, the techniques used in the embodiments are conventional methods well known to those skilled in the art.

[0029] Furthermore, 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 one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0030] Example 1: Screening of molecular marker combinations for Culter alburnus In this embodiment, 131 Culter alburnus were used as parents. Juvenile fish were obtained through natural reproduction. The fins of 300 juvenile fish were randomly sampled for whole-genome resequencing to obtain the original loci (original sequencing data). GWAS analysis was performed based on the original data, resulting in 2108 functional SNP loci. Based on the original loci, 10155 background SNP loci were obtained through screening, for a total of 12263 SNP loci.

[0031] The specific screening process includes the following steps: S1. Obtaining high-quality SNP sites: S11. Sample Collection: 45 Culter alburnus from Danjiangkou Reservoir, 40 from Taihu Lake, and 46 from Poyang Lake, totaling 131 fish, were collected and cultured together as broodstock at the Comprehensive Experimental Base of the Zhejiang Freshwater Fisheries Research Institute. Juvenile fish were obtained through natural reproduction in June, and DNA samples were randomly collected from the fins of 300 juvenile fish.

[0032] S12. Acquisition of Raw SNP Loci: DNA samples from the fins of 300 randomly selected juvenile fish were used for whole-genome resequencing. Raw reads were initially filtered and compared with the reference genome of *Culter alburnus*, yielding 9,939,714 raw SNP loci. Specifically, whole-genome resequencing of the DNA samples from the fins of the 300 juvenile fish was performed using the Illumina PE 150 platform, with a total sequencing depth of 3948.55 Gb, an average sequencing depth of 13.16 Gb per sample, and an average sequencing depth of approximately 12.5× per sample. The specific workflow involved constructing a sequencing library for each sample, and all DNA libraries were sequenced on the Illumina PE 150 platform.

[0033] The initial filtering conditions are as follows: 1) Sequencing fragments containing adapters (reads); 2) Low-quality reads (more than 50% of the reads have a quality value of Q≤5); 3) Reads containing more than 5% N (N indicates that the base information cannot be determined).

[0034] The process of comparing and screening the Culter alburnus reference genome was as follows: all (preliminarily filtered sequencing fragments) clean data were aligned to the Culter alburnus reference genome (version number GWHBOSX00000000.fa) sequence using BWA software; variant sites were detected and genotyped using GATK (v4.6.0.0) software; and SNP detection results were annotated using ANNORVAR software, resulting in 9,939,714 SNP sites.

[0035] S13. Quality control filtering was performed on the SNP sites, resulting in 6,353,900 high-quality SNP sites. The quality control filtration process is as follows: The obtained genotypic data (9,939,714 SNP loci) were quality-controlled using PLINK (v1.90b4) software. The filtering criteria were: deletion rate >10%, minor allele frequency (MAF) <0.05, and Hardy-Weinberg equilibrium test P <10. -6 Ultimately, 300 individuals and 6,353,900 SNPs were available for GWAS analysis.

[0036] S2, Functional site screening: S21. Obtain phenotypic information related to the economic traits of Culter alburnus from the juvenile fish used for DNA sample collection: phenotypic information includes the body length, body height, and weight of the above 300 Culter alburnus at 10 months of age, and the body length, body width, weight, condition factor, and specific growth rate at 18 months of age.

[0037] S22. Perform quality control on phenotypic information; remove samples whose phenotypic deviation exceeds three standard deviations.

[0038] S23. GWAS analysis was performed on high-quality SNP loci and phenotypic information after quality control; SNP loci related to the economic traits of Culter alburnus were obtained, and 2108 functional SNP loci were screened; specifically, GWAS analysis was performed using TASSEL software with a mixed linear model (MLM(K) model). Taking the weight phenotype of 18-month-old fish as an example, y = Xβ + Zμ + ε Where y is the phenotypic vector, i.e., the weight of 18-month-old Culter alburnus. X is the fixed effects matrix, and β is the fixed effects coefficient vector. Z is the random effects design matrix, and μ is the kinship random effects vector. ε is the residual vector. Principal component analysis was performed on the factors influencing the phenotypic profile, and the first five principal components were added as covariates to the model.

[0039] Genome-wide association analysis (GWAS) identified 502 SNP molecular markers associated with the weight trait of 18-month-old Culter alburnus.

[0040] Other SNP loci related to economic traits were obtained in a similar manner to those related to body weight. A total of 2911 functional SNP loci related to growth traits were obtained; SNP loci for which probes could not be designed at both ends were removed, resulting in 2108 functional SNP loci.

[0041] S3, Background Site Filtering: From 9,939,714 SNPs in 300 Culter alburnus resequencing data, background sites with read depth ≥10×, genotype deletion less than 10%, heterozygous genotype less than 20%, and minimum allele frequency greater than 0.05 were selected. SNP sites that could not be used to design probes at both ends were removed, resulting in 10,155 background SNP sites. These were then combined with 2,108 functional SNP sites to form 12,263 SNP sites.

[0042] The information on the 2108 functional SNP sites in this embodiment is shown in Table 2.

[0043] Table 2 .

[0044] As shown in Table 2: This embodiment includes 12,263 SNP loci, comprising 2,108 SNP loci related to economic traits of Culter alburnus and 10,155 background loci. The SNP loci related to economic traits include 142 loci related to body weight, 527 loci related to body length, 1,020 loci related to body height, 155 loci related to visceral weight, 114 loci related to body weight without visceral, 25 loci related to total length, 18 loci related to tail length, 6 loci related to condition factor (CF), and 218 loci related to gutted yield (GY).

[0045] Example 2: Fabrication of Culter alburnus 10K liquid phase chip The Culter alburnus 10K liquid phase chip includes a probe array for identifying genotypes at 12,263 SNP loci.

[0046] The purpose of this embodiment is to apply the 12263 SNP sites in Table 1 to probe design. Using GenoBaitsProbe Designer software, probes were designed based on the evaluation results of the upstream and downstream sequences of the target site. The evaluation mainly focused on the complexity of the upstream and downstream sequences, GC content, etc., prioritizing the placement of the target site in the middle of the probe. The designed probe was 110 bp in length. Generally, probe performance was comprehensively evaluated based on GC content (30%-70%), Tm value (60-80℃), and the number of alignments to the reference genome (≤5). The probe was 110 bp in length and contained a biotinylated DNA nucleotide sequence at its 5' end.

[0047] The distribution map of labeled chromosomes in the 10K liquid-phase microarray of *Culter alburnus* selected in this embodiment is shown below. Figure 1 The distribution statistics of the 10K liquid phase chip of Culter alburnus are shown in the figure below. Figure 2 As shown, the gene structure distribution diagram of the 10K liquid-phase chip for Culter alburnus is as follows: Figure 3 As shown.

[0048] Depend on Figures 1-3 It can be known that: Molecular markers in the 10K liquid-phase microarray of Culter alburnus were uniformly distributed on all 24 chromosomes of Culter alburnus. The average number of SNP markers in each chromosome was 511, ranging from 343 to 880. The vast majority of SNPs were located in introns and intergenic regions of the Culter alburnus genome.

[0049] Example 3: A kit comprising the Culter alburnus 10K liquid phase chip developed in Example 2 or comprising probes and / or primers for detecting 12,263 SNP sites.

[0050] Example 4: The method for genotyping 12263 SNP loci in Table 1 using the Culter albus 10K liquid phase chip developed in Example 2 includes the following steps: (1) Extraction of genomic DNA from Culter alburnus: Culter alburnus tail fin tissue was sampled according to standard procedures and genomic DNA was extracted using an animal genomic DNA extraction kit produced by Shijiazhuang Borui Biotechnology Co., Ltd. (2) DNA sample quality testing: The concentration of genomic DNA was detected using NanoDrop, ensuring that the DNA concentration was ≥10 ng / μL and 1.8 ≤ OD 260 / OD 280 ≤2.0, OD 260 / OD 230 For samples with a mass fraction of ≥2.0, agarose gel electrophoresis with a mass fraction of 1% is used for detection. The integrity of the DNA sample is determined by a gel imaging system to ensure that the main band of the sample is clear and intact.

[0051] (3) Liquid-phase chip testing: Library construction, target region capture and high-throughput sequencing of samples were performed using the standardized procedures and operations of Borui Biotechnology Co., Ltd. Step 31: DNA Fragmentation and End Repair PCR amplification system: 300 ng DNA; 2.6 μL terminal repair enzyme; 4 μL buffer; add nucleic acid-free water to a final volume of 20 μL. PCR amplification program: 37℃ for 20 min; 72℃ for 20 min.

[0052] Step 32: In the system from step 31, PCR is introduced into the adapter ligation system. PCR system: adapter 4 μL; ligase 2 μL; buffer 8 μL; nucleic acid-free water to bring the total to 20 μL; PCR amplification program: 22℃ for 60 min.

[0053] Step 33: Purify the product from the ligation system. 33.1 Add magnetic beads to the PCR product and shake well; 33.2 After standing at room temperature for 5 minutes, briefly centrifuge; 33.3 Place the PCR tube on a magnetic rack until the solution becomes clear; 33.4 Carefully remove the supernatant, making sure not to attract the magnetic beads; 33.5 Keep the PCR tube / plate in the magnetic rack, add 100 μL of 80% ethanol, and let stand at room temperature for 30 seconds; 33.6 Remove the supernatant, keep the PCR tube / plate in the magnetic rack, and leave at room temperature until the ethanol has completely evaporated.

[0054] Step 34: Perform PCR amplification using Culter alburnus genomic DNA as a template to obtain a DNA library. PCR amplification system: Add 10 μL of the mixture of PCR enzyme and buffer to the product from step 33; add 2 μL of sequencing barcode; and bring the total volume to 20 μL with nucleic acid-free water. PCR amplification program: 98℃ for 2 min for 1 cycle; (98℃ for 30 s, 65℃ for 30 s, 72℃ for 40 s) for 5 cycles; 72℃ for 4 min for 1 cycle.

[0055] Step 35: Purify the obtained DNA library. 35.1 Add 20 μl of GenoPrep DNA Clean Beads to the product obtained in step 34, vortex to mix, being careful not to generate gas during vortexing, let stand for 5 min, and then briefly centrifuge. 35.3 Place the PCR tubes / plates on a magnetic rack until the solution is clear, expecting to stand at room temperature for at least 3 minutes; 35.4 Remove the supernatant, making sure it does not attract the magnetic beads; 35.5 Keep the PCR tubes / plates in the magnetic rack and add 100 μL of 80% ethanol. Incubate at room temperature for 30 seconds; 35.6 Remove the supernatant, keep the PCR tube / plate in the magnetic rack, and leave at room temperature until the ethanol has completely evaporated; 35.7 Remove the PCR tube or plate from the magnetic rack, add 35 μL of Eulsion Buffer, vortex to mix, let stand for 5 min, and then briefly centrifuge. 35.8 Place the PCR tube on a magnetic rack until the solution becomes clear; 35.9 Transfer the supernatant to a new 0.2 ml low-adsorption PCR tube / plate.

[0056] Step 36: Mix equal amounts of the purified DNA library.

[0057] Step 37: Add the concentrated mixed library to the hybridization system and use the prepared probe for hybridization capture. PCR amplification system: 1.5-2.5 μg of well-mixed library; 300 ng of probe; add nucleic acid-free water to a final volume of 16 μl; PCR amplification program: 95℃ for 10 min (heated lid temperature 105℃); 65℃ for 2-4 h (heated lid temperature 75℃).

[0058] Step 38: Elute to remove unbound DNA 38.1 After hybridization is complete, open the PCR instrument lid, open the PCR tube lid, and transfer 16 μL of hybridization capture solution into the prepared magnetic beads; 38.2 Vortex oscillation for 10 seconds to fully mix, then instantaneously centrifuge; 38.3 Place the PCR tube into the PCR instrument, 65℃, 45min, with the hot cap at 75℃; 38.4 Centrifuge briefly every 12 minutes, shaking for 5 seconds. 38.5 Add 100 μL of preheated (65°C) elution buffer to each PCR tube; 38.6 Brief oscillation vortex for 5 seconds, centrifugal force for 5 seconds; 38.7 Place the PCR tube on the magnetic rack until the magnetic beads are completely separated from the solution; 38.8 Remove the supernatant with a pipette, retaining the magnetic beads, and place the PCR tube in a PCR instrument at 65°C; 38.9 Add 150 μL of preheated GenoBaits 1X Stringent Wash Buffer (65℃), and slowly pipette up and down 10 times to thoroughly mix the magnetic beads. After the last batch of samples is mixed, place it on the PCR instrument for 2 minutes; 38.10 Place the PCR tube on a magnetic rack until the magnetic beads are completely separated from the solution, then quickly remove the supernatant with a pipette; 38.11 Transfer the PCR tube from the magnetic rack, add 150 μL of room temperature elution buffer I, and vortex for 2 min; 38.12 Place the PCR tube on a magnetic rack until the magnetic beads are completely separated from the solution. Remove the supernatant using a pipette; 38.13 Transfer the PCR tube from the magnetic rack, add 150 μL of room temperature elution buffer II, and vortex for 1 min; 38.14 Place the PCR tube on a magnetic rack until the magnetic beads are completely separated from the solution. Remove the supernatant using a pipette; 38.15 Add 150 μL of room temperature elution buffer III and vortex for 30 seconds; 38.16 Place the PCR tube on a magnetic rack until the magnetic beads are completely separated from the solution. Remove the supernatant using a pipette; 38.17 Remove the test tube containing the captured DNA from the magnetic rack and add 20 μL of nuclease-free water. Gently pipette 10 times to ensure all beads are resuspended.

[0059] Step 39: Enrich the document library PCR system: DNA enriched on magnetic beads in step 38; 15 μL enzyme mixture; 1.2 μL primers; add nucleic acid-free water to a final volume of 30 μL; PCR amplification program: 98℃ for 45s for 1 cycle; (98℃ for 15s, 60℃ for 30s, 72℃ for 30s) for 13 cycles; 72℃ for 1min.

[0060] Step 40: Purify the enriched product to complete the preparation of the sequencing library. 40.1 Place the PCR tube on a magnetic rack until the solution becomes clear; 40.2 Transfer the supernatant to a new PCR tube; 40.3 Add 45 μL of magnetic beads to each reaction and shake to mix. 40.4 After standing at room temperature for 5 minutes, briefly centrifuge; 40.5 Place the PCR tube on a magnetic rack until the solution becomes clear; 40.6 Remove the supernatant using a pipette; 40.7 Keep the PCR tube on a magnetic rack and add 100 μL of 80% ethanol. Incubate at room temperature for 30 seconds; 40.8 Remove the supernatant using a pipette; 40.9 Keep the PCR tubes on a magnetic rack at room temperature until the ethanol evaporates completely; 40.10 Remove the PCR tube from the magnetic rack to room temperature, add 35 μL of elution buffer, and vortex to mix. After standing at room temperature for 5 min, briefly centrifuge. 40.11 Place the PCR tube on a magnetic rack until the solution becomes clear; 40.12 Transfer the supernatant to a new 0.2 mL low-adsorption tube, ensuring no magnetic beads are aspirated. The purified DNA library can be stored at -20°C until sequencing is required.

[0061] Step 41: Use equal-volume mixed sequencing libraries for high-throughput sequencing using the BGI T7 sequencer.

[0062] (4) Bioinformatics analysis: Use GATK software to analyze the mutation sites of the sequencing results and obtain the genotype of the target site; use IGV software to view the mutations of the sequencing results and the reference genome.

[0063] Of the 12 Culter alburnus samples tested (the parents of these 12 samples came from Danjiangkou), the samples were named DJK01, DJK02, DJK03, DJK04, DJK05, DJK06, DJK07, DJK08, DJK09, DJK10, DJK11, and DJK12. The SNP detection rate ranged from 98.47% to 98.76%, with an average detection rate of 98.65%. Figure 4 As shown, the cGPS detection results meet the development standards.

[0064] The 10K liquid phase chip for Culter alburnus can perform genotyping and DNA detection on Culter alburnus samples based on the above detection method.

[0065] Example 5: Application of 10K liquid phase chip in Culter alburnus population breeding 1) Collected the tail fins of 1319 Culter alburnus and extracted DNA; 2) Targeted capture sequencing of all individuals was performed using a 10K liquid phase array for Culter alburnus, yielding a total of 1216.80 Gb of raw data. After quality control using fastp (V0.20.0, parameters: -n 10 -q 20 -u 40), 1155.20 Gb of cleanbase was obtained. 3) Use BWA to align clean reads to the reference genome sequence (GWHBOSX00000000.fa) to obtain genotype data for each locus; 4) Analysis showed that the average detection rate of SNP loci in the samples was 98.68%, and the average heterozygosity rate was 38.17%. The GBLUP method was used to predict the genomic estimated breeding value (GEBV) of individuals for growth traits such as body weight and body length in Culter alburnus (see the distribution map of genomic breeding values ​​(body weight) for all individuals). Figure 5 The distribution map of all individual genome breeding values ​​(body length) is shown below. Figure 6 The model's prediction accuracy for weight and body length traits was 0.3247 and 0.3059, respectively, indicating that the 10K liquid-phase chip for Culter alburnus has high reliability in genome-wide selection breeding. Finally, individuals ranking in the top 5% based on their combined weight and body length breeding values ​​were selected as candidate parents for further breeding.

[0066] Example 6: Application of 10K liquid phase chip in the breeding of superior Culter alburnus varieties: The whole genome resequencing data of the Culter alburnus sample developed in Example 2 can be obtained using the 10K liquid phase chip for Culter alburnus. The 2108 SNP loci listed in Table 2 can be detected, and the detection rate of these 2108 SNP loci can be used to determine whether the economic traits of the tested Culter alburnus meet the requirements. For example: when the detection rate of 2108 SNP loci is above 99%, it means that the chromosome of the sample contains 2108 SNP loci, and the sample meets the economic trait requirements of the Culter alburnus; when the detection rate of 2108 SNP loci is less than 99%, it means that the chromosome of the sample only contains some of the 2108 SNP loci, and further analysis is needed to determine which loci are among the 2108 SNP loci and their corresponding traits. Based on the missing traits and the requirements of the breeding goals, it is determined whether the sample meets the requirements: if the missing trait does not affect the breeding goals, then it meets the requirements.

[0067] In a specific case: Assuming the breeding goal is to obtain Culter alburnus with a high meat content, one Culter alburnus can be selected and its data can be obtained using the Culter alburnus 10K liquid phase chip developed in Example 2. The 2108 SNP sites in Table 2 can be detected. If 99% of the 2108 SNP sites are detected, the Culter alburnus meets the requirements. If the detection rate does not meet the requirements, it is necessary to determine whether the missing sites are related to the meat content (body weight, eviscerated body weight, visceral weight, fatness, and evisceration rate) of the Culter alburnus. If they are not related, the requirements are still met; otherwise, the requirements are not met.

[0068] The descriptions in Examples 5 and 6 demonstrate that the 10K liquid phase chip for Culter alburnus of the present invention is applicable to the breeding and assisted breeding of Culter alburnus.

[0069] Example 7: Application of 10K liquid phase chip for detecting the economic characteristics of Culter alburnus Whole-genome resequencing data were obtained from 120 Culter alburnus samples. The 2108 SNP loci in the samples were detected using a 10K liquid phase microarray, with a detection rate exceeding 99.26%. This demonstrates that the 2108 SNP loci selected in this invention can be used for the detection of economic traits (body weight, body length, body height, visceral weight, eviscerated weight, total length, tail length, condition factor, and eviscerated percentage) in Culter alburnus.

[0070] Example 8: Application of 10K liquid phase chip in genome-wide association study of Culter alburnus Using the 10K liquid phase chip for Culter alburnus developed in Example 2, the genotype data of 131 Culter alburnus from Example 1 were obtained and filled using the genotype detection method in Example 4. Based on the mixed linear model, the economic traits of the 131 Culter alburnus were analyzed.

[0071] The association analysis results are shown in Table 2. It was determined that 2108 functional SNP sites were associated with the economic traits of Culter alburnus.

[0072] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0073] It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes to aid those skilled in the art and to facilitate understanding. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to size, without affecting the effectiveness and purpose of the invention, should still fall within the scope of the disclosed technical content. Furthermore, terms such as "upper," "lower," "left," "right," and "middle" used in this specification are merely for clarity and not intended to limit the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention's implementation.

Claims

1. The application of molecular marker combinations for Culter alburnus, characterized in that, Applications include genotyping of Culter alburnus, detection of economic traits in Culter alburnus, genome-wide association studies of Culter alburnus, and breeding or assisted breeding of Culter alburnus. Economic traits of Culter alburnus include body weight, body length, body height, visceral weight, eviscerated weight, total length, tail length, condition factor, and evisceration rate. The molecular marker combination includes 12,263 SNP sites. The physical locations of these 12,263 SNP sites were determined based on whole-genome sequence alignment of the *Culter alburnus* reference genome, with version number GWHBOSX00000000.fa. The specific physical location information of the 12,263 SNP sites is shown in Table 1 below. Table 1 。 2. The application of the molecular marker combination of Culter alburnus shown in Table 1 of claim 1 in a gene breeding chip, characterized in that, The gene breeding chip includes a liquid phase chip.

3. A 10K liquid phase chip for Culter alburnus, characterized in that, The genotyping of the liquid-phase chip includes the 12,263 SNP sites as described in claim 1.

4. The 10K liquid phase chip for Culter alburnus according to claim 3, characterized in that, It also includes probes designed based on gene sequences covering 12,263 SNP sites.

5. The 10K liquid phase chip for Culter alburnus according to claim 4, characterized in that, The probe sequence has a GC content of 30-70%, a probe length of 110 bp, a maximum upper limit of less than 5 specific similar fragments on the reference genome, and a maximum distance of less than 10 bp from the designed region.

6. The design method of the 10K liquid phase chip for Culter alburnus as described in any one of claims 3-5, characterized in that, Includes the following steps: S1. Obtaining high-quality SNP sites: S11. Sample collection: Using Culter alburnus from different regions as parents, juvenile fish are obtained through natural reproduction, and DNA samples of the juvenile fish are collected. S12. Obtaining raw SNP sites: The DNA sample is subjected to whole genome resequencing, raw reads are initially filtered, and the Culter alburnus reference genome is compared and screened to obtain raw SNP sites; S13. Perform quality control filtering on the original SNP sites to obtain high-quality SNP sites; S2, Functional site screening: S21. Obtain phenotypic information related to the economic traits of Culter alburnus from the juvenile fish used for DNA sample collection. S22. Perform quality control on the phenotypic information; S23. Perform GWAS analysis on the high-quality SNP loci and the phenotypic information after quality control; obtain SNP loci related to the economic traits of Culter alburnus, and screen to obtain 2108 functional SNP loci; S3, Background Site Filtering: From the SNP sites obtained through comparison and screening of the Culter alburnus reference genome, 10,155 background SNP sites were obtained, which were combined with 2,108 functional SNP sites to form 12,263 SNP sites; S4. Probe Design: A probe sequence designed to detect 12,263 SNP sites.

7. The application of the Culter alburnus 10K liquid phase chip as described in any one of claims 3-5 in the detection of Culter alburnus genotype.

8. The application of the 10K liquid phase chip for Culter alburnus as described in any one of claims 3-5 in detecting the economic characteristics of Culter alburnus, characterized in that, The economic traits include body weight, body length, body height, visceral weight, eviscerated weight, total length, tail length, fatness, and evisceration rate.

9. The application of the Culter alburnus 10K liquid phase chip as described in any one of claims 3-5 in Culter alburnus breeding, in Culter alburnus genome-wide association analysis, or in Culter alburnus assisted breeding.

10. A reagent kit, characterized in that, Includes the Culter alburnus 10K liquid phase chip as described in any one of claims 3-5, or includes probes and / or primers for detecting the 12263 SNP sites shown in Table 1.

11. The use of the kit as described in claim 10 in detecting the genotype of Culter alburnus.

12. The application of the kit as described in claim 10 in detecting economic traits of Culter alburnus, characterized in that, The economic traits include body weight, body length, body height, visceral weight, eviscerated weight, total length, tail length, fatness, and evisceration rate.

13. The application of the kit as described in claim 10 in the breeding of Culter alburnus, or in the genome-wide association study of Culter alburnus, or in the assisted breeding of Culter alburnus.

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