Oncorhynchus mykiss whole genome SNP molecular marker combination, liquid phase breeding chip and application thereof

Abstract

The invention belongs to the technical field of gene chip technology and fish molecular breeding, and relates to a rainbow trout whole genome SNP molecular marker combination, a liquid phase breeding chip and application thereof. The invention constructs an oncorhynchus mykiss SNP liquid phase breeding chip with 50K site density. The chip comprises an oncorhynchus mykiss whole genome SNP molecular marker combination composed of 51508 SNP molecular markers. The liquid-phase breeding chip is high in labeling density and high in automation suitability, and can support high-throughput detection; in addition, the applicability and universality of the chip in rainbow trout groups with different genetic backgrounds are improved, so that the chip can be widely applied to research work in multiple aspects such as germplasm resource identification, genetic diversity evaluation, whole-genome selective breeding, whole-genome association analysis and genetic map construction of rainbow trout at home and abroad; the technical requirements of rainbow trout cross-region breeding and accurate breeding are met.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene chip and fish molecular breeding, and particularly relates to a rainbow trout whole genome SNP molecular marker combination, a liquid breeding chip and application thereof. BACKGROUND

[0002] Rainbow trout (Oncorhynchus mykiss) has become a representative variety of cold water aquaculture fish in the world due to its excellent growth rate and outstanding muscle quality. In recent years, China has made key technical breakthroughs in the design and construction of factory circulating water aquaculture facilities and large steel structure deep sea aquaculture net cages, providing an equipment foundation for the large-scale development of marine aquaculture rainbow trout industry. However, at present, the seed of the rainbow trout aquaculture industry in China mostly depends on western countries such as the United States, Canada, Denmark and Norway, and high-quality seed is sold in the form of "all female" or "triploid", and the source is strictly controlled, which to a large extent restricts the large-scale development of the marine aquaculture industry of salmonids in China. Therefore, how to use efficient modern molecular breeding techniques and tools to quickly breed high-quality rainbow trout seed has become the key to breaking the problem of lack of good seed of salmonids in China.

[0003] Genomic selection breeding is a new method of genetic breeding using high-density markers covering the whole genome, which has the advantages of short cycle and high accuracy, and has been applied to the good seed breeding research of many important aquatic economic species, and its breeding effect is much better than that of traditional population breeding and family breeding technology. Salmonids have experienced a specific fourth whole genome duplication event, resulting in a genome size about 2-4 times that of ordinary bony fish. The high sequencing cost and huge computing resource consumption have become an important limiting factor for the widespread development of salmonid genomic selection breeding. Breeding chip is a recognized important modern molecular breeding tool, which can quickly and accurately genotype the massive SNP sites of germplasm resources, and has a low unit detection cost. In addition, breeding chip accelerates the development and application of whole genome selection in the breeding of aquatic economic species, and brings revolutionary changes to the field of genetic breeding of aquatic species.

[0004] Currently, two rainbow trout breeding chips have been developed worldwide, but both have limitations: the first rainbow trout breeding chip was developed by the USDA in 2014, which contains 57,501 SNP sites. However, due to technical limitations at the time, the chip contains about 20,000 redundant SNP sites, affecting its efficiency in typing rainbow trout SNP genotypes. The second rainbow trout SNP breeding chip (667K) was released by the French National Institute for Agricultural Research in 2021. Due to the high density of SNP sites on the chip and its development for European and American wild and breeding populations of rainbow trout, its applicability to Asian rainbow trout breeding populations is poor, limiting its application in scientific research of rainbow trout in China.

[0005] Based on this, the present application develops a 50K density of rainbow trout SNP liquid breeding chip, most of which are derived from the main breeding areas of rainbow trout in Shandong, Liaoning, Gansu and other domestic regions, and integrates genetic resources from multiple countries in Europe and the United States. It not only ensures the adaptability of the breeding chip to local rainbow trout populations, but also takes into account the genetic information of foreign rainbow trout breeding and wild populations, which is helpful for the analysis of the genetic basis of important economic traits and the excavation of excellent gene resources of rainbow trout at home and abroad. SUMMARY

[0006] In order to overcome the limitations of the above-mentioned rainbow trout breeding chip in terms of site redundancy, cost control and population adaptability, the present application proposes a rainbow trout whole genome SNP molecular marker combination, a liquid breeding chip and its application. The present application constructs a 50K site density of rainbow trout liquid SNP breeding chip. The marker density of the liquid breeding chip is high, and the automation adaptability is strong, which can support high-throughput detection. In addition, the present application also improves the applicability and universality of the chip in different genetic background of rainbow trout populations, so that it can be widely used in the identification of germplasm resources, genetic diversity assessment, whole genome selection breeding, whole genome association analysis and genetic map construction of rainbow trout at home and abroad. It meets the technical needs of rainbow trout cross-regional breeding and precision breeding.

[0007] The technical scheme of the present application is:

[0008] The present application provides a rainbow trout whole genome SNP molecular marker combination, which comprises 51508 SNP molecular markers. The site information of the SNP molecular markers is shown in Table 1 of the rainbow trout reference genome USDA_OmykA_1.1.

[0009] Table 1 Site information of SNP molecular markers

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[0106] The present application also provides a rainbow trout whole genome SNP targeted capture probe set, wherein the probes of the probe set are 5' end biotin group modified DNA nucleotide sequences synthesized according to the SNP molecular marker of claim 1.

[0107] The probe set is used for specific recognition or detection of the above-mentioned SNP molecular marker combination.

[0108] The present application provides a rainbow trout SNP liquid breeding chip, comprising the above-mentioned SNP molecular marker combination.

[0109] The rainbow trout (Oncorhynchus mykiss) SNP liquid breeding chip provided by the application comprises 51,508 SNP sites. The SNP sites are developed based on 495 high-depth whole genome resequencing data at home and abroad, cover the core functional regions of the whole genome of the rainbow trout, and the reference position is based on the rainbow trout reference genome version (USDA_OmykA_1.1) published by NCBI.

[0110] The rainbow trout SNP liquid breeding chip is a high-throughput SNP liquid chip, adopts targeted capture sequencing technology (cGPS) to realize high-precision genotyping of the SNP sites, 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] Further, the liquid breeding chip is loaded with the probe group.

[0112] The application provides a rainbow trout whole genome 50K SNP liquid breeding chip, comprising 51,508 SNP sites, located in the reference genome USDA_OmykA_1.1 of the rainbow trout, and named as "Rainbow No. 1".

[0113] Compared with the rainbow trout breeding chip developed by the US Department of Agriculture and the French National Agricultural Research Institute, the rainbow trout liquid breeding chip developed by the application has stronger applicability in the study of domestic rainbow trout breeding populations, especially for the growth rate and muscle quality of rainbow trout in seawater environment, can promote the genetic selection process of domestic seawater breeding rainbow trout, and helps to break through the bottleneck problem that the current rainbow trout breeding depends on imported seed sources and technology. At the same time, the development experience of the chip can also provide a technical paradigm for molecular marker assisted breeding of other important aquatic economic species such as large yellow croaker and prawn. With the coordinated development of deep sea aquaculture and intelligent fishery equipment, the chip is expected to be deeply integrated with intelligent breeding system, and provide key technical support for promoting the rainbow trout industry in China to a hundred billion level.

[0114] The application also provides a construction method of the rainbow trout SNP liquid breeding chip, comprising the following steps:

[0115] I. Screening of 50K liquid chip SNP sites of rainbow trout

[0116] (1) According to the whole genome resequencing data of 495 rainbow trout individuals at home and abroad, the SNP site set on the genome of all samples is constructed by aligning to the reference genome (USDA_OmykA_1.1) of the rainbow trout, for selection of target SNP sites;

[0117] (2) To ensure that the developed rainbow trout liquid breeding chip has high typing quality and wide population applicability, after preliminary identification of SNP sites, further screening is carried out according to the following standards: the missing rate (Miss rate) is less than 0.1, that is, the missing proportion of the site in the sample is not more than 10%; there should be no tandem repeat sequence in the adjacent region of the target SNP site to avoid probe design failure or non-specific hybridization; the heterozygosity rate (Heter rate) is less than or equal to 0.4 to exclude unstable or misjudgment-prone sites; the minimum allele frequency (MAF) is greater than or equal to 0.1 to ensure that the site has good polymorphism in the population; and the sequencing depth of the site is greater than or equal to 10x to ensure the accuracy and reliability of site detection;

[0118] (3) The candidate SNP sites to be selected are checked for typing, and repeated items are removed, and SNP sites with correct typing and evenly distributed on the chromosome are retained; and the present application integrates the SNP functional sites related to the sea-cultured traits of rainbow trout identified independently, and introduces the functional markers reported by domestic and foreign research institutions which are closely related to important economic traits of rainbow trout, thereby enhancing the practical value of the chip in trait association analysis and breeding selection.

[0119] Further, the cultured traits include any one or several of sea water body length, sea water body height, sea water body weight, sea water weight gain rate, sea water specific growth rate, back muscle intermuscular fat content, abdominal muscle intermuscular fat content or salinity tolerance.

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

[0121] In the present application, after obtaining a high-quality SNP candidate site set of rainbow trout, further probe design and screening are carried out:

[0122] (1) First, the typing accuracy of the preliminarily screened SNP sites is checked, and by comparing the reference alleles with the NCBI rainbow trout reference genome (USDA_OmykA_1.1), sites with repetition, unclear positioning or inconsistency with the reference sequence are removed, and effective SNP sites with good typing consistency are retained for subsequent design;

[0123] (2) In the retained effective SNP sites, further follow the design principle of "uniform distribution on the chromosome", to ensure that each chromosome uniformly covers the SNP sites in the physical position, to avoid that the sites are concentrated in a specific area or have too many empty areas, thereby improving the representativeness and breeding coverage ability of the chip;

[0124] (3) the screened SNP sites and the upstream and downstream sequences thereof are submitted to a probe design platform for comprehensive scoring, and the evaluation contents include: complexity (such as low complexity sequence or repetitive sequence) of the upstream and downstream sequences of the target site, and central positioning of the target SNP site in the probe sequence; finally, DNA nucleotide sequences (probe sequences) with better specificity and stability are screened, and biotin modification is added to the 5' end thereof, which is referred to as the rainbow trout 50K SNP site probe.

[0125] The application provides the use of the SNP molecular marker combination described above.

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

[0127] (2) application in identification of rainbow trout germplasm resources;

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

[0129] (4) application in whole genome correlation analysis of rainbow trout;

[0130] (5) application in breeding of rainbow trout;

[0131] (6) application in diagnosis of genetic defect diseases of rainbow trout;

[0132] (7) application in construction of genetic map of rainbow trout.

[0133] The application further provides the use of the rainbow trout SNP liquid breeding chip described above.

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

[0135] (2) application in identification of rainbow trout germplasm resources;

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

[0137] (4) application in whole genome correlation analysis of rainbow trout;

[0138] (5) application in breeding of rainbow trout;

[0139] (6) application in diagnosis of genetic defect diseases of rainbow trout;

[0140] (7) application in construction of genetic map of rainbow trout.

[0141] Further, the breeding is molecular marker assisted breeding, including whole genome selection breeding.

[0142] The application has the following beneficial effects:

[0143] (1) The rainbow trout liquid phase chip SNP sites 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 a wide range of sample population sources and full species coverage, and can be widely used in the detection of rainbow trout species materials at home and abroad.

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

[0145] This rainbow trout SNP liquid-phase breeding array boasts advantages such as rich trait-associated loci, strong versatility, low cost, high efficiency, high locus polymorphism, and uniform genomic distribution. It facilitates the elucidation of the genetic basis of important economic traits and the discovery of superior genetic resources in rainbow trout both domestically and internationally, making it suitable for widespread application in large-scale rainbow trout genetic improvement and germplasm evaluation. Furthermore, this rainbow trout SNP liquid-phase breeding array is more applicable to research on rainbow trout breeding populations in China, particularly for seawater growth rate and muscle quality, potentially advancing the genetic selection and breeding of improved rainbow trout strains in my country's marine aquaculture.

[0146] (3) The rainbow trout 50K SNP liquid-phase breeding chip provided by the present invention is constructed based on targeted capture sequencing technology and features good locus representation, high polymorphism, and uniform chromosomal distribution of loci. It is a precise and efficient molecular breeding chip. Targeted capture sequencing technology not only enables accurate SNP genotyping of rainbow trout, but also can be used for a variety of research needs, including germplasm identification, genetic diversity assessment, whole-genome selective breeding, whole-genome association analysis, and genetic map construction of wild and breeding rainbow trout populations at home and abroad. Furthermore, the target SNP loci can be adjusted by directly adding or removing probes, offering greater flexibility than solid-phase microarrays.

[0147] (4) Compared with traditional resequencing methods, the rainbow trout 50K SNP liquid-phase breeding chip provided by the present invention has the outstanding advantages of convenient operation, rapid completion of SNP typing, significantly improved detection efficiency, and reduced detection costs. The chip of the present invention is based on high-throughput sequencing technology. This detection method has high detection throughput and a large amount of data output at one time, which can cover the detection of nearly 1,000 materials simultaneously. It is also applicable to mainstream second-generation sequencing platforms such as Illumina and MGI, and has platform wide adaptability.

[0148] (5) The rainbow trout 50K SNP liquid breeding chip of the present application can be used for the application of germplasm resource identification, genetic diversity evaluation, whole genome selection breeding, whole genome association analysis and genetic map construction of domestic and foreign rainbow trout wild and breeding populations. BRIEF DESCRIPTION OF DRAWINGS

[0149] Figure 1 The figure shows the schematic diagram of the rainbow trout liquid breeding chip genotyping process.

[0150] Figure 2 The figure shows the SNP site annotation information statistics of the rainbow trout SNP liquid breeding chip provided by the present application and the distribution on the rainbow trout chromosome.

[0151] Figure 3 The figure shows the PCA principal component clustering effect comparison of the rainbow trout SNP liquid breeding chip provided by the present application and the resequencing data.

[0152] Figure 4 The figure shows the comparison of the phylogenetic tree of the rainbow trout SNP liquid breeding chip provided by the present application and the resequencing data.

[0153] Figure 5 The figure shows the comparison of the population genetic structure results of the rainbow trout SNP liquid breeding chip provided by the present application and the resequencing data. DETAILED DESCRIPTION

[0154] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0155] In order to further understand the present application, the present application will be further described in combination with the drawings and embodiments.

[0156] The experimental methods in the following embodiments are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following embodiments can be obtained from commercial channels, unless otherwise specified.

[0157] Example 1

[0158] A construction method of a rainbow trout SNP liquid breeding chip

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

[0160] To ensure the representativeness and universality of the SNP sites selected in the rainbow trout breeding chip development, the present application integrates the whole genome resequencing data of multiple representative rainbow trout populations at home and abroad, a total of 495 samples, to provide data basis for the screening of high-quality SNP sites and the construction of the chip.

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

[0162] The present application collects and integrates a total of 299 individuals from the main domestic rainbow trout breeding areas and typical breeding populations abroad. The domestic rainbow trout samples are from the core rainbow trout breeding areas of Gansu, Liaoning, Shandong, Xinjiang, Hubei, Hebei and Henan, etc. The foreign rainbow trout samples are from rainbow trout breeding lines in Denmark and Spain. Fin tissue of each rainbow trout individual is collected, whole genome DNA is extracted through standard procedures, and further WGS whole genome resequencing is performed to provide data basis for the subsequent screening of high-quality SNP sites.

[0163] (2) Integration of public resequencing data of foreign populations

[0164] The present application also introduces public resequencing data of representative breeding populations of foreign rainbow trout, a total of 196 samples, covering rainbow trout breeding populations from Oregon, California, Idaho and Alaska in the United States, rainbow trout breeding populations from British Columbia in Canada, Ontario in Canada and rainbow trout breeding lines from AquaGen company in Norway, the relevant data is from NCBI SRA database (Bioproject PRJNA402066, PRJNA386519), all of which are high-coverage resequencing data with good representativeness of foreign rainbow trout populations.

[0165] 2. Whole genome resequencing of rainbow trout populations at home and abroad

[0166] The 299 rainbow trout sample materials collected at home and abroad are subjected to whole genome resequencing, the specific steps including:

[0167] (1) High-quality genomic DNA is extracted by magnetic bead method;

[0168] (2) The qualified DNA samples are subjected to PCR amplification to form sequencing library;

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

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

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

[0172] The present application is based on whole genome resequencing data of 495 rainbow trout individuals at home and abroad, and SNPs are detected and screened by bioinformatics tools, and the analysis process is as follows:

[0173] (1) The original resequencing data is subjected to quality control by using fastp, and low-quality reads and adapter contamination are removed;

[0174] (2) The BWA software is used to align the high-quality sequencing reads after quality control to the rainbow trout reference genome provided by NCBI (version number: GCF_013265735.2_USDA_OmykA_1.1), and the default parameters are used for alignment, and a SAM format file is generated, and the SAMtools is used to convert it to a BAM format file, and sorting and indexing are performed at the same time;

[0175] (3) The Picard tool is used to remove duplicate reads generated by PCR from the above alignment results to obtain a de-duplicated BAM file;

[0176] (4) Based on the de-duplicated BAM file, the GATK (Genome Analysis Toolkit) software is used for single nucleotide polymorphism (SNP) site detection (SNP calling), and the GATK HaplotypeCaller module is used to output a GVCF format file; then the GenotypeGVCFs module is used to merge the GVCF file and generate a full-sample VCF file;

[0177] (5) The original SNP result file is subjected to preliminary filtering, and the following filtering standards are set to obtain high-quality SNP sites: heterozygosity ≤0.4; deletion rate <0.1; minimum allele frequency ≥0.1; sequencing depth ≥10x. Finally, a VCF format file containing high-quality SNP site information of 495 samples is obtained, a total of 7,773,979 SNP sites are detected, which are used as candidate SNP site data sets for subsequent chip development.

[0178] 4. Screening and determination of background sites of rainbow trout SNP liquid breeding chip

[0179] On the basis of initially obtaining 7,773,979 SNP sites, in order to ensure the design efficiency of the probe and the balance of genome coverage in subsequent chip development, the present application further screens 46,042 SNP sites for breeding chip background coverage. The screening steps are as follows:

[0180] (1) Removal of redundant and clustered sites: First, the chromosome distribution of all SNPs was analyzed, and SNP sites with close spacing and redundant functional annotations in dense regions were removed to avoid probe interference and reduced capture efficiency;

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

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

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

[0184] 5. Screening of SNP sites with high differentiation index 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, the present invention systematically screened SNP sites associated with important phenotypes and population differentiation based on the Fst (Fixation Index) analysis strategy at the whole genome level.

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

[0187] (2) Eighteen domestic and foreign rainbow trout farming populations were used as units. Each population was combined with the remaining 17 populations and then Fst was compared between each population. Highly differentiated SNP sites with Fst>0.3 were screened out. After probe design and removal of failed sites, a total of 4,313 candidate functional sites with strong correlation with population differentiation were screened out to enhance the genetic discrimination ability of the chip for rainbow trout populations from different geographical origins.

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

[0189] To enhance the application value of the liquid breeding chip in the important economic traits of rainbow trout, the present application identifies functional SNP sites related to key economic traits by genomic selection (GS) and genome-wide association study (GWAS) of the research team.

[0190] (1) In terms of GWAS, the present application uses 8 sets of phenotype data of 180 rainbow trout samples from Gansu, Liaoning and Shandong cultured in seawater environment, and high-quality SNP genotype information, and the traits are 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 and salt tolerance. Using gemma software, based on mixed linear model (MLM), genome-wide association analysis is performed on 8 traits, and covariates such as kinship matrix, gender, sequencing batch and principal component are introduced for correction to reduce background interference, and the threshold is set to 0.05 / SNP total site number. After probe design screening, 67 SNP functional sites are finally obtained for inclusion in the rainbow trout SNP liquid breeding chip.

[0191] (2) In terms of GS, the present application also uses the above-mentioned 8 traits of seawater culture phenotype information of rainbow trout samples and high-quality SNP genotype information to construct multiple statistical models for prediction accuracy evaluation, including GBLUP, BayesA, BayesB, BayesCπ and BayesLASSO. Among them, the BayesB model shows the highest prediction ability in multiple traits and is selected as the optimal GS model. According to the marker effect value and stability output by the BayesB model, SNP functional sites with stable contribution to 8 traits are selected. After probe adaptability and specificity filtering, 621 SNP sites are finally retained for chip development.

[0192] 7. Literature-related functional sites of rainbow trout economic traits

[0193] To further enhance the functional marker coverage of the rainbow trout SNP liquid breeding chip in core economic traits, the present application systematically collects and integrates functional SNP sites related to rainbow trout economic traits published in recent years at home and abroad, covering multiple representative traits, including:

[0194] (1) Low oxygen 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;

[0196] (3) Fillet color (DOI: https: / / doi.org / 10.3390 / genes13081331), 46;

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

[0198] (5) Comparison of anadromous and landlocked (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] The present application obtains 593 SNP sites with clear functional annotation or phenotype correlation evidence by co-integration of the above-mentioned literatures. Subsequently, all candidate sites are evaluated for sequence adaptability and specificity by a chip probe design platform, including but not limited to technical parameters such as sequence complexity, GC content, Tm value, alignment uniqueness, etc. Finally, 63 sites are selected, which have both functional representation and probe design feasibility, and are included in the rainbow trout SNP liquid breeding chip development.

[0202] All SNP sites in steps 4-7 above are integrated to form a rainbow trout 50K SNP liquid breeding chip, and the number of sites is finally 51,508 (Table 1). The distribution of SNP sites in chromosomes is shown in Figure 2 .

[0203] 8. Development of rainbow trout 50K SNP liquid breeding chip

[0204] The selected 51,508 candidate SNP sites are synthesized into liquid phase capture probes by Huazhi Biotechnology Co., Ltd., and the precise positioning sequencing typing technology (cGPS) based on target region genome sequence liquid phase capture is used to form the system of rainbow trout liquid chip.

[0205] cGPS is based on an optimized thermodynamic stability algorithm model to design probes for different target regions of the genome sequence, use synthetic specific probes to capture and enrich multiple different target sequences located at different genomic positions by liquid phase hybridization, then construct sequencing library and high-throughput sequencing of the captured and enriched target genomic sequence, so as to obtain the genotype of all SNP sites in the target region, such as Figure 1 shown.

[0206] Example 2

[0207] Polymorphism analysis of SNP sites on the rainbow trout SNP liquid breeding chip

[0208] In this embodiment, the polymorphism and chromosome distribution of the SNP sites contained in the constructed rainbow trout SNP liquid breeding chip were analyzed.

[0209] First, the PLINK software was used to calculate the polymorphism information content (Polymorphism Information Content, PIC) of the SNP sites screened by the chip. The actual measured population genotype frequency data of the chip was used to calculate the PIC value of each site, and the PIC of all SNPs was statistically summarized to obtain an average PIC value of 0.32, which belongs to a moderate polymorphism level (0.25 < PIC < 0.5). Subsequently, the chromosome distribution of the SNP sites on the rainbow trout reference genome (NCBI number: GCF_013265735.2_USDA_OmykA_1.1) was statistically analyzed. The specific operation is as follows:

[0210] (1) Using the site positioning information in the chip design process, the chromosome number and physical position (unit: bp) of each SNP site were extracted;

[0211] (2) The number of all SNP sites on the 32 chromosomes was counted using R language, and a SNP distribution map was drawn;

[0212] (3) Further calculate the physical distance between adjacent SNP sites to obtain the average spacing of SNP sites on each chromosome;

[0213] (4) Integrating the data of 32 chromosomes, the average spacing of SNP sites in this chip was 43.3Kb, indicating that the distribution of sites in the chip on the genome was relatively uniform.

[0214] The analysis results show that this chip has good genome coverage and moderate polymorphism level, and is suitable for subsequent genomic selection and breeding research. The SNP site chromosome distribution map is shown in Figure 2

[0215] Example 3 ​

[0216] Performance Verification of Rainbow Trout SNP Liquid-Phase Breeding Chip

[0217] To verify the typing stability and applicable range of the rainbow trout SNP liquid-phase breeding chip of the present invention, this example used the chip constructed in Example 1 to perform SNP typing detection on 80 rainbow trout individuals from home and abroad.

[0218] The sample sources were as follows: 80 rainbow trout, 56 of which were from breeding colonies in Gansu, Liaoning, Shandong, Xinjiang, and Sichuan, China; and 24 from commercially farmed colonies in Spain, Poland, and Denmark. To assess reproducibility, 16 of these samples were randomly selected for technical replicates, yielding a total of 96 SNP genotyping data.

[0219] 1. DNA Extraction and Quality Control

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

[0221] 2. Chip detection

[0222] SNP genotyping was performed using a rainbow trout SNP liquid-phase breeding array. DNA from each sample was digested, labeled, amplified, and hybridized, followed by hybridization signal capture using an automated liquid-phase array platform.

[0223] 3. Data Processing and Quality Control

[0224] Raw data were normalized for signal intensity, background corrected, and genotyped using AxiomAnalysis Suite software. Software parameters were set to default, and a GT call quality score (QC) was generated for each SNP locus. The overall SNP call rate (call rate) was calculated using the formula: number of successfully typed SNPs per sample ÷ total number of SNPs on the array × 100%. Genotype concordance was calculated by comparing the concordance of GT values ​​for each locus across technical replicates. Analysis results showed that across 96 samples, the SNP call rate ranged from 99.44% to 99.91%, with an average of 99.76%. Among 16 pairs of technical replicates, the genotype concordance ranged from 99.48% to 99.71%, with an average of 99.58%.

[0225] The above results show that the rainbow trout SNP liquid-phase breeding chip has a high site success rate and good repeat consistency, the typing results are stable and reliable, and it is suitable for the detection of SNP markers in rainbow trout 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 array and resequencing data

[0228] This example aims to compare the consistency of the results of rainbow trout SNP breeding array and resequencing genetic structure analysis for the same rainbow trout sample.

[0229] The 51,508 high-quality SNP loci from the rainbow trout liquid-phase SNP breeding array designed by this invention were used to analyze the population genetic structure of 495 rainbow trout samples from China and abroad. To verify the reliability of the rainbow trout liquid-phase array typing data, the present invention used 495 whole-genome resequencing data from the same batch of rainbow trout samples as a control, and used the same analysis process to analyze the SNP data of the population genetic structure. The software and parameters used for genetic structure analysis are as follows:

[0230] (1) Principal component analysis of the chip typing data was performed using GCTA software to reveal the genetic variation and main differentiation trends among the samples. First, the 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) MEGA software was used to construct a phylogenetic tree (Neighbor-Joining method) based on the genetic distance matrix to analyze the kinship 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 converted to a format supported by MEGA (.meg). Then, the Neighbor-Joining (NJ) method was used to construct the phylogenetic tree. 1000 bootstrap replicates were performed to assess the stability of the tree structure. Finally, the kinship and clustering patterns between samples from different populations were visualized.

[0232] (3) Population structure analysis was performed using ADMIXTURE software. First, the SNP data were converted into binary BED format (plink--make-bed). ADMIXTURE was used to run the structure inference model in the range of K = 2 to K = 10, and the optimal K value was selected using the CV error (cross-validation error). Finally, the genetic components of each sample at 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 results of population genetic structure analysis based on the typing data of the rainbow trout SNP breeding chip of the present invention are highly consistent with the analysis results obtained based on the 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 evaluation and molecular breeding research.

[0235] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, and modifications made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A rainbow trout genome-wide SNP molecular marker combination, characterized in that: The SNP molecular marker combination includes 51,508 SNP molecular markers, and the position information of the SNP molecular markers in the rainbow trout reference genome USDA_OmykA_1.1 is shown in Table 1.

2. A rainbow trout genome-wide SNP targeted capture probe set, characterized in that: The probe of the probe group is a DNA nucleotide sequence modified with a biotin group at the 5' end synthesized by the SNP molecular marker according to claim 1; The probe group is used to specifically identify or detect the SNP molecular marker combination according to claim 1.

3. A rainbow trout SNP liquid phase breeding chip, characterized in that: Comprising the SNP molecular marker combination according to claim 1.

4. The rainbow trout SNP liquid phase breeding chip according to claim 3, characterized in that The liquid-phase breeding chip is loaded with the probe group according to claim 2.

5. The SNP molecular marker combination according to claim 1 has any of the following uses: (1) Application in rainbow trout genotyping; (2) Application in the identification of rainbow trout germplasm resources; (3) Application in analysis of rainbow trout genetic diversity; (4) Application in genome-wide association analysis of rainbow trout; (5) Application in rainbow trout breeding; (6) Application in the diagnosis of rainbow trout genetic defects; (7) Application in the construction of rainbow trout genetic map.

6. The rainbow trout SNP liquid phase breeding chip according to claim 3 has any of the following uses: (1) Application in rainbow trout genotyping; (2) Application in the identification of rainbow trout germplasm resources; (3) Application in analysis of rainbow trout genetic diversity; (4) Application in genome-wide association analysis of rainbow trout; (5) Application in rainbow trout breeding; (6) Application in the diagnosis of rainbow trout genetic defects; (7) Application in the construction of rainbow trout genetic map.

7. The use according to claim 5 or 6, characterized in that The breeding is molecular marker assisted breeding, including whole genome selection breeding.

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