A rainbow trout whole genome SNP molecular marker combination, liquid breeding chip and application thereof

Abstract

This invention belongs to the fields of gene chip technology and fish molecular breeding technology, and relates to a rainbow trout whole-genome SNP molecular marker combinatorial, a liquid-phase breeding chip, and their applications. This invention constructs a rainbow trout SNP liquid-phase breeding chip with a density of 50K loci. This chip includes a rainbow trout whole-genome SNP molecular marker combinatorial consisting of 51,508 SNP molecular markers. This liquid-phase breeding chip has high marker density, strong automation adaptability, and can support high-throughput detection. Furthermore, this invention improves the applicability and versatility of the chip in rainbow trout populations with different genetic backgrounds, enabling it to be widely applied to various research needs in rainbow trout germplasm resource identification, genetic diversity assessment, whole-genome selection breeding, genome-wide association analysis, and genetic map construction both domestically and internationally, meeting the technical requirements for cross-regional breeding and precision selection of rainbow trout.

Description

Technical Field

[0001] This invention belongs to the fields of gene chip technology and fish molecular breeding technology, specifically involving a rainbow trout whole genome SNP molecular marker combination, liquid phase breeding chip and its application. Background Technology

[0002] Rainbow trout (Oncorhynchus mykiss), with its excellent growth rate and superior muscle quality, has become a representative species of cold-water aquaculture fish worldwide. In recent years, my country has achieved key technological breakthroughs in the design and construction of factory-scale recirculating aquaculture systems and large-scale steel-structured deep-sea aquaculture cages, providing the equipment foundation for the large-scale development of the marine rainbow trout aquaculture industry. However, currently, my country's rainbow trout aquaculture industry relies heavily on seedlings from Western countries such as the United States, Canada, Denmark, and Norway. Their high-quality seedlings are sold in the form of "all-female" or "triploid," with strict control over the source of the seedlings, which significantly restricts the large-scale development of my country's salmon and trout marine aquaculture industry. Therefore, how to utilize efficient modern molecular breeding technologies and tools to rapidly cultivate high-quality rainbow trout seedlings has become crucial to overcoming the problem of the scarcity of superior salmon and trout breeds in my country.

[0003] Genome selection breeding is a novel method for genetic selection using high-density markers covering the entire genome. It boasts advantages such as short cycle time and high accuracy, and has been applied to the breeding research of many important aquatic economic species, with breeding results far superior to traditional population selection and family selection techniques. Salmon and trout, having undergone a specific fourth genome duplication event, have genomes approximately 2-4 times larger than those of common bony fishes. The resulting high sequencing costs and enormous computational resource consumption have become significant limiting factors for the widespread implementation of genome selection breeding in salmon and trout. Breeding microarrays are recognized as important modern molecular breeding tools, enabling rapid and accurate genotyping of massive SNP loci in germplasm resources with relatively low unit detection costs. Furthermore, breeding microarrays have accelerated the development and application of genome-wide selection in the breeding of aquatic economic species, bringing revolutionary changes to the field of aquatic genetic breeding.

[0004] Currently, two rainbow trout breeding chips have been developed worldwide, but both have limitations: The world's first rainbow trout breeding chip was developed by the USDA in 2014, containing 57,501 SNP loci. However, due to technological limitations at the time, the chip contained approximately 20,000 redundant SNP loci, affecting its genotyping efficiency in rainbow trout SNPs. The world's second rainbow trout SNP breeding chip (667K) was released by the French National Institute for Agricultural Research in 2021. Due to its excessively high SNP locus density and its development for wild and breeding rainbow trout populations in Europe and America, its applicability to Asian rainbow trout breeding populations is poor, limiting its application in rainbow trout scientific research in my country.

[0005] Based on this, the present invention develops a 50K density rainbow trout SNP liquid phase breeding chip, in which most of the SNP loci are derived from major rainbow trout breeding areas in China, such as Shandong, Liaoning, and Gansu, and integrate genetic resources from multiple European and American countries. This ensures the adaptability of the breeding chip to local rainbow trout populations while taking into account the genetic information of rainbow trout breeding and wild populations abroad, which helps to analyze the genetic basis of important economic traits of rainbow trout at home and abroad and to explore superior gene resources. Summary of the Invention

[0006] To overcome the limitations of existing rainbow trout breeding chips in terms of site redundancy, cost control, and population adaptability, this invention proposes a rainbow trout genome-wide SNP molecular marker combination, a liquid-phase breeding chip, and its applications. This invention constructs a rainbow trout liquid-phase SNP breeding chip with a site density of 50K sites. This liquid-phase breeding chip features high marker density, strong automated adaptability, and supports high-throughput detection. Furthermore, this invention improves the applicability and versatility of the chip in rainbow trout populations with different genetic backgrounds, enabling its widespread application in various research areas both domestically and internationally, including rainbow trout germplasm resource identification, genetic diversity assessment, genome-wide selection breeding, genome-wide association analysis, and genetic map construction. This meets the technical requirements for cross-regional breeding and precision selection of rainbow trout.

[0007] The technical solution of this invention is:

[0008] This invention provides a whole-genome SNP molecular marker combinatorial for rainbow trout, which contains 51,508 SNP molecular markers. The location information of the SNP molecular markers in the rainbow trout reference genome USDA_OmykA_1.1 is shown in Table 1.

[0009] Table 1. Site information of SNP molecular markers

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[0106] The present invention also provides a rainbow trout whole genome SNP targeted capture probe set, wherein the probes of the probe set are DNA nucleotide sequences with biotinylated groups at the 5' end synthesized according to the SNP molecular markers described in claim 1;

[0107] The probe set is used to specifically identify or detect the SNP molecular marker combination described above.

[0108] This invention provides a rainbow trout SNP liquid-phase breeding chip, comprising the SNP molecular marker combination described above.

[0109] The rainbow trout (Oncorhynchus mykiss) SNP liquid-phase breeding chip provided by this invention includes 51,508 SNP loci. These SNP loci were developed based on 495 high-depth whole-genome resequencing data from domestic and international sources, covering the core functional regions of the rainbow trout genome. Their reference positions are based on the rainbow trout reference genome version (USDA_OmykA_1.1) published by NCBI.

[0110] The rainbow trout SNP liquid phase breeding chip is a high-throughput SNP liquid phase chip that uses targeted capture sequencing (cGPS) technology to achieve high-precision genotyping of SNP loci. It has the advantages of high sensitivity, high throughput, and strong adaptability, and is suitable for rainbow trout populations with different genetic backgrounds at home and abroad.

[0111] Furthermore, the liquid-phase breeding chip is loaded with the probe set described above.

[0112] This invention provides a liquid-phase breeding chip for rainbow trout with 50K SNPs in the whole genome, including 51,508 SNP loci located in the rainbow trout reference genome USDA_OmykA_1.1, and named "Rainbow Core No. 1".

[0113] The rainbow trout liquid-phase breeding chip developed in this invention is more applicable to domestic rainbow trout breeding population research than those developed by the US Department of Agriculture and the French National Institute for Agricultural Research (INRA). It is particularly effective in improving the growth rate and muscle quality of rainbow trout in marine environments, and can advance the genetic selection and breeding of superior rainbow trout breeds in my country's marine aquaculture, helping to overcome the current bottleneck of reliance on imported seed sources and technology in rainbow trout breeding. Furthermore, the development experience of this chip can provide a technological paradigm for molecular marker-assisted breeding of other important aquatic economic species such as large yellow croaker and shrimp. With the synergistic development of deep-sea aquaculture and intelligent fisheries equipment, this chip is expected to be deeply integrated with intelligent aquaculture systems, providing key technological support for propelling my country's rainbow trout industry towards a scale of tens of billions of yuan.

[0114] This invention also provides a method for constructing the rainbow trout SNP liquid-phase breeding chip described above, comprising the following steps:

[0115] I. Screening of SNP sites for rainbow trout on a 50K liquid-phase microarray

[0116] (1) Based on the whole genome resequencing data of 495 rainbow trout individuals from home and abroad, the reference genome of rainbow trout (USDA_OmykA_1.1) was compared, and a set of SNP sites on all sample genomes was constructed for the selection of target SNP sites.

[0117] (2) To ensure that the developed rainbow trout liquid-phase breeding chip has high genotyping quality and wide population applicability, after the initial identification of SNP sites, further screening was carried out according to the following criteria: Miss rate < 0.1, that is, the proportion of missing sites in the sample does not exceed 10%; there should be no tandem repeat sequences in the adjacent region of the target SNP site to avoid probe design failure or hybridization nonspecificity; Heter rate ≤ 0.4 to exclude unstable or easily misjudged sites; Minimum allele frequency (MAF) ≥ 0.1 to ensure that the site has good polymorphism in the population; site sequencing depth ≥ 10x to ensure the accuracy and reliability of site detection;

[0118] (3) The candidate SNP sites to be selected are genotyped and verified, duplicates are eliminated, and SNP sites that are correctly genotyped and evenly distributed on chromosomes are retained. Furthermore, this invention integrates SNP functional sites related to rainbow trout marine aquaculture traits that have been independently identified, and introduces functional markers that are closely related to important economic traits of rainbow trout that have been reported by domestic and foreign research institutions, thereby enhancing the practical value of the chip in trait association analysis and breeding selection.

[0119] Furthermore, the aquaculture traits include any one or more of the following: seawater body length, seawater body height, seawater body weight, seawater weight gain rate, seawater specific growth rate, back muscle intramuscular fat content, abdominal muscle intramuscular fat content, or salinity tolerance.

[0120] II. Design and preparation of rainbow trout 50K SNP site probes

[0121] In this invention, after obtaining a high-quality set of candidate SNP sites for rainbow trout, probe design and screening are further carried out:

[0122] (1) First, the genotyping accuracy of the preliminary SNP sites was checked. By comparing the reference allele with the NCBI rainbow trout reference genome (USDA_OmykA_1.1), sites with duplication, unclear location or inconsistency with the reference sequence were removed, and valid SNP sites with good genotyping consistency were retained for subsequent design.

[0123] (2) Among the retained effective SNP sites, the design principle of "uniform chromosome distribution" is further followed to ensure that each chromosome covers the SNP sites evenly in physical location, avoiding the sites being densely concentrated in a specific area or having too many empty areas, thereby improving the representativeness and breeding coverage of the chip.

[0124] (3) The selected SNP sites and their upstream and downstream sequences are submitted to the probe design platform for comprehensive evaluation. The evaluation includes the complexity of the upstream and downstream sequences of the target site (such as low-complexity sequences or repetitive sequences) and the central location of the target SNP site in the probe sequence. Finally, DNA nucleotide sequences (probe sequences) with better specificity and stability are selected and biotin is added to their 5' end, which are called rainbow trout 50K SNP site probes.

[0125] This invention provides the above-described SNP molecular marker combinations for use as described in any of the following:

[0126] (1) Application in rainbow trout genotyping detection;

[0127] (2) Application in rainbow trout germplasm resource identification;

[0128] (3) Application in genetic diversity analysis of rainbow trout;

[0129] (4) Application in genome-wide association studies of rainbow trout;

[0130] (5) Application in rainbow trout breeding;

[0131] (6) Application in the diagnosis of genetic defects in rainbow trout;

[0132] (7) Application in the construction of rainbow trout genetic maps.

[0133] The present invention also provides that the rainbow trout SNP liquid-phase breeding chip described above has any of the following uses:

[0134] (1) Application in rainbow trout genotyping detection;

[0135] (2) Application in rainbow trout germplasm resource identification;

[0136] (3) Application in genetic diversity analysis of rainbow trout;

[0137] (4) Application in genome-wide association studies of rainbow trout;

[0138] (5) Application in rainbow trout breeding;

[0139] (6) Application in the diagnosis of genetic defects in rainbow trout;

[0140] (7) Application in the construction of rainbow trout genetic maps.

[0141] Furthermore, the breeding is molecular marker-assisted breeding, including genome-wide selection breeding.

[0142] The beneficial effects of this invention are:

[0143] (1) The SNP sites of the rainbow trout liquid phase chip provided by the present invention are composed of whole genome resequencing data of 495 rainbow trout individuals from domestic and foreign rainbow trout populations. It has the advantages of wide sample population sources and full variety coverage, and can be widely used for the detection of rainbow trout varieties at home and abroad.

[0144] (2) The rainbow trout 50K SNP liquid phase breeding chip provided by this invention covers SNP loci related to important economic traits of rainbow trout screened by domestic and foreign research institutes. These mainly include: SNP functional loci related to key economic traits such as seawater growth rate, muscle quality and high salt tolerance of rainbow trout screened by our research team based on genome selection breeding, population genetic differentiation research and genome-wide association analysis; and high-quality SNP functional loci related to traits such as hypoxia tolerance, high temperature tolerance and resistance to columnar flavobacterium identified by foreign research institutions.

[0145] This rainbow trout SNP liquid-phase breeding chip boasts advantages such as abundant trait-associated loci, strong versatility, low cost, high efficiency, high locus polymorphism, and uniform genome distribution. It facilitates the analysis of the genetic basis of important economic traits in rainbow trout both domestically and internationally, and the discovery of superior gene resources. It is suitable for widespread application in large-scale rainbow trout genetic improvement and germplasm evaluation. Furthermore, this rainbow trout SNP liquid-phase breeding chip is more applicable to domestic rainbow trout breeding population research, especially for marine growth rate and muscle quality, which can promote the genetic selection and breeding of superior rainbow trout breeds in my country's marine aquaculture industry.

[0146] (3) The rainbow trout 50K SNP liquid-phase breeding chip provided by this invention is constructed based on targeted capture sequencing technology. It has the characteristics of good site representativeness, high polymorphism, and uniform distribution of sites on chromosomes. It is a precise and efficient molecular breeding chip. Not only can it achieve precise SNP genotyping of rainbow trout through targeted capture sequencing technology, but the results can also be used for various research needs such as germplasm resource identification, genetic diversity assessment, whole-genome selection breeding, whole-genome association analysis, and genetic map construction of wild and breeding populations of rainbow trout at home and abroad. Furthermore, the target SNP sites can be adjusted by directly adding or removing probes, which has better flexibility compared with solid-phase chips.

[0147] (4) Compared with traditional resequencing methods, the rainbow trout 50K SNP liquid-phase breeding chip provided by this invention has significant advantages in terms of convenient operation, rapid SNP genotyping, greatly improved detection efficiency, and reduced detection costs. The chip of this invention is based on high-throughput sequencing technology. This detection method has high throughput and produces a large amount of data at once, capable of simultaneously covering the detection of nearly a thousand materials. It is also suitable for mainstream second-generation sequencing platforms such as Illumina and MGI, demonstrating broad platform adaptability.

[0148] (5) The rainbow trout 50K SNP liquid phase breeding chip of the present invention can be used for the identification of germplasm resources, assessment of genetic diversity, whole genome selection breeding, whole genome association analysis and construction of genetic maps of wild and breeding populations of rainbow trout at home and abroad. Attached Figure Description

[0149] Figure 1 A schematic diagram of the rainbow trout liquid phase breeding chip genotyping process is shown.

[0150] Figure 2 The statistical information on SNP loci annotation in the rainbow trout SNP liquid-phase breeding chip provided by the present invention and their distribution on the rainbow trout chromosome are shown.

[0151] Figure 3 The paper presents a comparison of the PCA principal component clustering effects of the rainbow trout SNP liquid-phase breeding chip and resequencing data provided by this invention.

[0152] Figure 4 The diagram shows a comparison between the phylogenetic tree of the rainbow trout SNP liquid-phase breeding chip and the resequencing data provided by this invention.

[0153] Figure 5 The diagram shows a comparison of the population genetic structure results of the rainbow trout SNP liquid-phase breeding chip and resequencing data provided by this invention. Detailed Implementation

[0154] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0155] To further understand the present invention, it will be further described in conjunction with the accompanying drawings and embodiments.

[0156] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0157] Example 1

[0158] A method for constructing a liquid-phase breeding chip for rainbow trout SNP

[0159] 1. Collection and construction of rainbow trout germplasm resources

[0160] To ensure the representativeness and universality of the SNP sites selected in the development of rainbow trout breeding chips, this invention integrates whole-genome resequencing data from multiple representative rainbow trout populations at home and abroad, totaling 495 samples, providing a data foundation for the screening of high-quality SNP sites and the construction of chips.

[0161] (1) Collection of rainbow trout population samples and acquisition of resequencing data at home and abroad

[0162] This invention collected and integrated 299 individuals from major rainbow trout farming areas in China and typical breeding populations abroad. Domestic rainbow trout samples came from core rainbow trout farming areas including Gansu, Liaoning, Shandong, Xinjiang, Hubei, Hebei, and Henan. International rainbow trout samples were from rainbow trout farming lines in Denmark and Spain. Fin tissue was collected from each individual rainbow trout, and whole-genome DNA was extracted using standard procedures. Further whole-genome resequencing (WGS) was performed to provide a data foundation for subsequent screening of high-quality SNP loci.

[0163] (2) Integration of international public resequencing data

[0164] This invention also incorporates public resequencing data from representative rainbow trout breeding populations abroad, totaling 196 samples. These samples cover rainbow trout breeding populations from Oregon, California, Idaho, and Alaska in the United States, as well as rainbow trout breeding populations from British Columbia and Ontario in Canada, and rainbow trout farming lines from AquaGen in Norway. The relevant data are sourced from the NCBI SRA database (Bioproject PRJNA402066, PRJNA386519), and are all high-coverage resequencing data, demonstrating good representativeness of foreign rainbow trout populations.

[0165] 2. Whole-genome resequencing of rainbow trout populations both domestically and internationally

[0166] Whole-genome resequencing was performed on 299 rainbow trout samples collected from both domestic and international sources. Specific steps included:

[0167] (1) High-quality genomic DNA was extracted using the magnetic bead method;

[0168] (2) DNA samples that have passed quality inspection are amplified by PCR to form sequencing libraries;

[0169] (3) After the library passes the test, high-throughput sequencing is performed using 150bp paired end reads on the BGIDNBSEQ-T7 platform.

[0170] (4) The average sequencing depth of each sample is about 10x, and the average amount of raw sequencing data obtained per rainbow trout sample is 24Gb.

[0171] 3. Detection of SNP loci in rainbow trout populations at home and abroad

[0172] This invention is based on whole-genome resequencing data from 495 rainbow trout individuals from both domestic and international sources. Bioinformatics tools were used to detect and screen for SNPs. The analysis process is as follows:

[0173] (1) Use FastP to perform quality control on the raw resequencing data to remove low-quality reads and adapter contamination;

[0174] (2) The high-quality sequencing reads after quality control were aligned to the rainbow trout reference genome (version number: GCF_013265735.2_USDA_OmykA_1.1) provided by NCBI using BWA software. The alignment was performed using the default parameters to generate a SAM format file. The SAM tools were then used to convert the SAM file to a BAM format file, and the reads were sorted and indexed.

[0175] (3) Use Picard to remove duplicate reads generated by PCR from the above alignment results to obtain the deduplicated BAM file;

[0176] (4) Based on the deduplicated BAM file, the GATK (Genome Analysis Toolkit) software was used to detect single nucleotide polymorphism (SNP) sites (SNP calling). The GATKHaplotypeCaller module was used to output GVCF format files. Then, the GenotypeGVCFs module was used to merge the GVCF files and generate the full sample VCF file.

[0177] (5) The original SNP result files were initially filtered, and the following filtering criteria were set to obtain high-quality SNP loci: heterozygosity ≤ 0.4; deletion rate < 0.1; minimum allele frequency ≥ 0.1; sequencing depth ≥ 10x. Finally, a VCF format file containing high-quality SNP locus information from 495 samples was obtained, and a total of 7,773,979 SNP loci were detected, which will serve as the candidate SNP locus dataset for subsequent microarray development.

[0178] 4. Screening and determination of background loci in rainbow trout SNP liquid phase breeding microarray

[0179] Based on the initial acquisition of 7,773,979 SNP loci, to ensure the design efficiency of probes and the balance of genome coverage in subsequent microarray development, this invention further screened for 46,042 SNP loci for background coverage in breeding microarrays. The screening steps are as follows:

[0180] (1) Removal of redundant and clustered sites: First, perform chromosome distribution analysis on all SNPs and remove SNP sites in dense regions that are too close together and have redundant functional annotations to avoid probe interference and reduced capture efficiency.

[0181] (2) Balancing functional and neutral region coverage: Based on SnpEff annotation results, SNPs covering relatively neutral regions such as intergenic, intronic, and non-coding transcript regions are preferentially retained as the basic framework for background genetics.

[0182] (3) Optimize the uniform distribution of chromosomes: Set an equally spaced window (20kb) for each chromosome, and select 1-2 representative SNP sites that meet the quality standards in each window to ensure the uniform distribution of the selected SNP sites throughout the genome and avoid bias towards specific regions.

[0183] Finally, 46,042 SNP sites were selected and retained as the background marker site set for the chip, which was used to construct a high-coverage and highly versatile rainbow trout SNP liquid-phase chip framework.

[0184] 5. Screening of highly differentiated SNP sites among rainbow trout populations at home and abroad

[0185] To improve the applicability of rainbow trout SNP breeding chips in populations with different genetic backgrounds, this invention uses a genome-wide Fst (Fixation Index) analysis strategy to systematically screen SNP loci associated with important phenotypes and population differentiation.

[0186] (1) For the three key economic traits of rainbow trout, namely seawater growth rate, muscle quality and salt tolerance, the top 20% of individuals with the best and worst performance in each trait were compared. According to the different groups of rainbow trout, each SNP locus of all samples in each group was calculated (single point calculation) with the parameter --weir-fst-pop. Finally, the samples were filtered according to the threshold of Fst>0.3. After removing the failed design sites after probe design, a total of 402 candidate functional loci strongly related to the traits were screened out.

[0187] (2) Taking 18 domestic and foreign rainbow trout farming populations as units, each population was merged with the other 17 populations and then the pairwise Fst comparison was performed to screen out highly differentiated SNP sites with Fst>0.3. After probe design and removal of design failure sites, a total of 4,313 candidate functional sites with strong population differentiation correlation were screened out to enhance the chip's ability to distinguish the genetics of rainbow trout populations from different geographical sources.

[0188] 6. Functional SNP loci of rainbow trout economic traits obtained based on GS and GWAS analysis

[0189] To enhance the application value of liquid-phase breeding chips in important economic traits of rainbow trout, this invention combines the functional SNP loci associated with key economic traits identified by our research team through Genomic Selection (GS) and Genome-Wide Association Study (GWAS).

[0190] (1) Regarding GWAS, this invention used phenotypic data of eight traits and high-quality SNP genotypic information from 180 rainbow trout samples cultured in marine environments from Gansu, Liaoning, and Shandong provinces. The traits were marine body length, marine body height, marine body weight, marine weight gain rate, marine specific growth rate, dorsal muscle intramuscular fat content, abdominal muscle intramuscular fat content, and salinity tolerance. Using Gemma software, genome-wide association analysis was performed on each of the eight traits based on a mixed linear model (MLM). Covariates such as kinship matrix, sex, sequencing batch, and principal components were introduced for correction to reduce background interference. The threshold was set to 0.05 / total number of SNP loci. After probe design and screening, 67 functional SNP loci were finally obtained and incorporated into the rainbow trout SNP liquid-phase breeding chip.

[0191] (2) Regarding GS, this invention also uses the marine aquaculture phenotypic information of the above-mentioned rainbow trout samples for 8 traits, along with high-quality SNP genotypic information, to construct multiple statistical models for predictive accuracy evaluation, including GBLUP, BayesA, BayesB, BayesCπ, and BayesLASSO. Among them, the BayesB model showed the highest predictive ability among multiple traits and was selected as the optimal GS model. Based on the marker effect value and stability output by the BayesB model, SNP functional sites that have a stable contribution to the 8 traits were screened. Finally, after probe adaptability and specificity filtering, 621 SNP sites were retained for chip development.

[0192] 7. Collection of functional loci related to economic traits in rainbow trout from relevant literature

[0193] To further enhance the functional marker coverage capability of rainbow trout SNP liquid-phase breeding chips for core economic traits, this invention systematically collects and integrates publicly published functional SNP sites related to economic traits of rainbow trout in recent years, covering multiple representative traits, specifically including:

[0194] (1) Hypoxia tolerance (DOI:https: / / doi.org / 10.1016 / j.aquaculture.2022.739068), 15;

[0195] (2) High temperature tolerance (DOI:https: / / doi.org / 10.1111 / eva.13240), 15 samples;

[0196] (3) Color of meat slices (DOI:https: / / doi.org / 10.3390 / genes13081331), 46;

[0197] (4) Muscle hardness (DOI:https: / / 10.3389 / fgene.2019.00386), 365 samples;

[0198] (5) Comparison between migratory and landlocked types (DOI:https: / / doi.org / 10.3389 / fgene.2022.795850), 24;

[0199] (6) Carcass traits (DOI:https: / / 10.3389 / fgene.2016.00203), 100;

[0200] (7) Flavobacterium columnare resistance (DOI:https: / / doi.org / 10.1016 / j.aquaculture.2022.738332), 28.

[0201] This invention integrates 593 SNP loci with clear functional annotations or phenotypic association evidence from the aforementioned literature. Subsequently, using a chip probe design platform, sequence fit and specificity were evaluated for all candidate loci, including but not limited to sequence complexity, GC content, Tm value, alignment uniqueness, and other technical parameters. Finally, 63 loci were selected and retained, which combine functional representativeness and probe design feasibility, and were incorporated into the development of rainbow trout SNP liquid-phase breeding chip.

[0202] All SNP loci from steps 4-7 above were integrated to form a liquid-phase breeding chip for rainbow trout 50K SNPs. The final number of loci was 51,508 (Table 1). The distribution of SNP loci on the chromosome is shown in the figure. Figure 2 .

[0203] 8. Development of a 50K SNP liquid-phase breeding chip for rainbow trout

[0204] The 51,508 candidate SNP sites selected were synthesized using liquid-phase capture probes by Huazhi Biotechnology Co., Ltd., and a rainbow trout liquid-phase chip system was formed using precise localization sequencing and genotyping technology (cGPS) based on liquid-phase capture of target region genome sequences.

[0205] Based on the optimized thermodynamic stability algorithm model, cGPS designs probes for genomic sequences in different target regions, uses synthetic specific probes to perform liquid-phase hybridization capture enrichment on multiple different target sequences located at different genomic positions, and then constructs a sequencing library and performs high-throughput sequencing on the captured and enriched target genomic sequences, so as to obtain the genotypes of all SNP sites within the target region, such as Figure 1 shown. <00005​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0216] Performance verification of rainbow trout SNP liquid phase breeding chip

[0217] To verify the typing stability and applicability of the rainbow trout SNP liquid-phase breeding chip of the present invention, this embodiment uses the chip constructed in Example 1 to perform SNP typing detection on 80 rainbow trout individuals from both domestic and international sources.

[0218] The sample sources are as follows: a total of 80 rainbow trout, of which 56 samples came from breeding populations in Gansu, Liaoning, Shandong, Xinjiang, and Sichuan provinces of China; and 24 samples came from commercial aquaculture populations in Spain, Poland, and Denmark. To assess the repeatability of genotyping, 16 trout were randomly selected from the above samples for technical replication testing, resulting in 96 SNP genotyping data.

[0219] 1. DNA extraction and quality control

[0220] After extracting DNA from fish fins or muscle tissue, the DNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer, with OD260 / 280 controlled between 1.8 and 2.0; DNA integrity was then detected by 1% agarose gel electrophoresis to ensure the absence of degraded bands.

[0221] 2. Chip testing

[0222] SNP genotyping was performed using a liquid-phase breeding chip for rainbow trout. Each sample used 200 ng of DNA, which was digested, labeled, amplified, and hybridized. Hybridization signals were then captured using an automated liquid-phase chip platform.

[0223] 3. Data Processing and Quality Control

[0224] Raw data were normalized for signal intensity, corrected for background, and genotype determined using Axiom Analysis Suite software. Default software parameters were used. The GT call quality score (QC) was output for each SNP locus, and the overall SNP call rate was calculated. The locus call rate was calculated as: (Number of successfully genotyped SNPs per sample ÷ Total number of SNPs on the microarray) × 100%. Genotype concordance was calculated by comparing the GT values ​​of each locus in technical replicates to determine the concordance rate. The analysis showed that in 96 samples, the SNP call rate ranged from 99.44% to 99.91%, with an average of 99.76%. In 16 pairs of technical replicates, the genotype concordance rate ranged from 99.48% to 99.71%, with an average of 99.58%.

[0225] The above results indicate that the rainbow trout SNP liquid phase breeding chip has a high site success rate and good repeatability consistency, and the genotyping results are stable and reliable. It is suitable for the detection of rainbow trout SNP markers from different regions and strains, and has good prospects for promotion and application.

[0226] Example 4

[0227] Comparative analysis of the genetic structure of rainbow trout populations at home and abroad based on rainbow trout SNP breeding chips and resequencing data

[0228] This embodiment aims to compare the consistency of results from rainbow trout SNP breeding chips and resequencing genetic structure analysis for the same rainbow trout samples.

[0229] The population genetic structure of 495 rainbow trout samples from both domestic and international sources was analyzed using 51,508 high-quality SNP loci from the rainbow trout liquid-phase SNP breeding chip designed in this invention. To verify the reliability of the rainbow trout liquid-phase chip genotyping data, this invention used 495 whole-genome resequencing data from the same batch of rainbow trout samples as controls, and performed population genetic structure analysis on their SNP data using the same analytical workflow. The software and parameters used for genetic structure analysis are shown below:

[0230] (1) Principal component analysis was performed on the chip genotyping data using GCTA software to reveal the genetic variation and main differentiation trends among the samples. First, a quality-controlled PLINK format file (.bed / .bim / .fam) was input, and the scores of the samples on the first two principal components (PC1 and PC2) were exported. The genetic distribution and clustering trends among the samples were visualized using R language.

[0231] (2) A phylogenetic tree was constructed based on the genetic distance matrix using MEGA software (Neighbor-Joining method) to analyze the phylogenetic relationships between different samples. First, the IBS (Identity by State) genetic distance matrix between samples was calculated using the PLINK tool. The generated distance matrix was then converted to a format supported by MEGA (.meg). Then, the Neighbor-Joining (NJ) method was used to construct the phylogenetic tree. 1000 bootstrap repetitions were performed to evaluate the stability of the tree structure. Finally, the phylogenetic relationships and clustering patterns between samples from different populations were visualized.

[0232] (3) The population structure analysis was performed using ADMIXTURE software. First, the SNP data was converted to binary BED format (plink-make-bed). ADMIXTURE was used to run the structure inference model in the range of K=2 to K=10. Then, the optimal K value was selected by using the CV error (cross-validation error). Finally, the genetic components of each sample under different K values ​​were plotted and displayed to determine the population differentiation trend.

[0233] The results are as follows Figures 3 to 5 As shown, the population genetic structure analysis results based on the genotyping data of the rainbow trout SNP breeding chip of this invention are highly consistent with the analysis results obtained based on whole genome resequencing data.

[0234] The above results indicate that the SNP sites selected in the rainbow trout SNP breeding chip have good polymorphism, representativeness, and population structure resolution capabilities, and can effectively replace whole genome resequencing data for large-scale germplasm resource assessment and molecular breeding research.

[0235] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, alterations, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rainbow trout whole-genome SNP-targeting capture probe set, characterized in that, The probes in the probe set are DNA nucleotide sequences with a biotinylate group modified at the 5' end, synthesized based on SNP molecular markers. The SNP molecular marker combination contains 51,508 SNP molecular markers, and the location information of the SNP molecular markers in the rainbow trout reference genome USDA_OmykA_1.1 is shown in Table 1. The probe set is used to specifically identify or detect the SNP molecular marker combination.

2. A rainbow trout SNP liquid-phase breeding chip, characterized in that, The liquid-phase breeding chip is loaded with the probe set as described in claim 1.

3. The probe set according to claim 1 has the use described in any one of the following: (1) Application in rainbow trout genotyping detection; (2) Application in the identification of rainbow trout germplasm resources; (3) Application in genetic diversity analysis of rainbow trout; (4) Application in genome-wide association studies of rainbow trout; (5) Application in rainbow trout breeding; (6) Application in the construction of rainbow trout genetic maps.

4. The rainbow trout SNP liquid-phase breeding chip according to claim 2 has any of the following uses: (1) Application in rainbow trout genotyping detection; (2) Application in the identification of rainbow trout germplasm resources; (3) Application in genetic diversity analysis of rainbow trout; (4) Application in genome-wide association studies of rainbow trout; (5) Application in rainbow trout breeding; (6) Application in the construction of rainbow trout genetic maps.

5. The use according to claim 3 or 4, characterized in that, The breeding is molecular marker-assisted breeding, including whole-genome selection breeding.

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