SNP site combination for largemouth bass genotyping and application thereof

Abstract

The application provides a SNP site combination for largemouth bass genotyping and application thereof. The SNP site combination comprises 50000 SNP sites, and the positions and base information of the 50000 SNP sites on chromosomes are shown in Table 3. The whole genome liquid phase chip of largemouth bass provided by the application can quickly and accurately detect the genotype of a sample to be detected, the average detection rate of the SNP sites and the average consistency rate of the sample genotypes are as high as 99.95%, and the whole genome liquid phase chip of largemouth bass has the characteristics of high capture specificity, low cost, flexible sites and the like, is suitable for trait selection and improvement, gene mining, kinship identification, genetic diversity analysis and the like of largemouth bass, and can effectively improve the selection efficiency and precision of a good strain of largemouth bass, and is favorable for cultivating a new variety of largemouth bass which is more suitable for market demand and more competitive.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular detection, and particularly relates to a SNP site combination for large-mouth bass genotyping and application thereof. BACKGROUND

[0002] Aquatic molecular assisted breeding is a key technological innovation in the field of modern aquaculture, which relies on advanced molecular biology methods to precisely assist aquatic breeding work. In practical application, by in-depth analysis of the genome of aquatic animals, researchers can quickly lock the molecular markers closely related to important economic traits such as growth rate, disease resistance, and stress resistance. With these markers, individuals with excellent traits can be precisely selected when aquatic animals are still in the seedling or even embryo stage, greatly shortening the long cycle of traditional breeding and significantly improving breeding efficiency. At the same time, molecular assisted breeding can be used to clearly analyze the genetic diversity of germplasm resources, accurately identify various aquatic germplasms, and effectively protect rare and endangered species. In addition, in the process of hybrid breeding, molecular markers can be used to scientifically select parents with large genetic differences and strong complementarity, efficiently predict hybrid vigor, and make each cross more targeted and successful, so as to continuously cultivate high-quality varieties with fast growth, excellent quality, and strong resistance for the aquaculture industry, and powerfully promote the aquaculture industry to a new journey of high yield, high quality, and sustainable development.

[0003] Liquid chip technology, also known as suspension array liquid chip technology, is an advanced high-throughput molecular detection method based on a microsphere suspension system. It couples microspheres with different color codes to nucleic acid, protein, and other types of biological molecule probes. In a liquid environment, these coupled probes can specifically bind to target molecules. After the reaction is complete, the results of the binding reaction can be quickly and accurately detected and analyzed using equipment such as a flow cytometer, enabling efficient and accurate determination of a large number of biological molecules. It plays an important role in biomedical, agricultural breeding, and especially aquatic breeding. In genetic diversity assessment, it can simultaneously detect multiple genetic loci in a large number of aquatic animal individuals, quickly and accurately analyze the genetic differences within and between populations, and provide precise data support for the rational planning of aquatic germplasm resource protection and utilization. For example, in the study of various fish populations, liquid chip technology can detect a large number of single nucleotide polymorphism (SNP) markers, clearly presenting the population genetic structure and assisting in the development of scientific germplasm management strategies. In molecular marker-assisted selection, liquid chip technology can screen a large number of molecular markers closely linked to important economic traits such as growth rate, disease resistance, and stress resistance. For example, in shrimp breeding, by detecting disease-resistant molecular markers, potential disease-resistant individuals can be accurately selected at an early stage, greatly shortening the breeding cycle and improving breeding efficiency, and cultivating more market-competitive new shrimp varieties. In addition, in the sex identification of aquatic animals, liquid chip technology can accurately identify sex-determining gene markers, which is helpful for the rational planning of breeding density and mode for some aquatic species whose sex difference significantly affects economic value, such as some fish and crustaceans, and further improves economic efficiency.

[0004] Micropterus salmoides, commonly known as California bass or largemouth bass, belongs to Perciformes, Percoidei, Centrarchidae, and Micropterus. Since its introduction in the 1970s, the production of Micropterus salmoides has shown a significant growth trend, with an annual production of over 800,000 tons nationwide. The new varieties "Youyu No. 1" and "Youyu No. 3" have been bred using traditional group selection techniques, and have improved in growth rate and deformity rate. However, long-term self-reproduction and inbreeding have led to increased deformity rate, decreased germplasm, and frequent diseases. As an important freshwater fish in China, the improvement of Micropterus salmoides germplasm and the breeding of superior varieties are of great significance to the high-quality development of the aquaculture industry. Therefore, the development of a whole-genome SNP liquid chip for Micropterus salmoides is urgently needed to improve the breeding efficiency of Micropterus salmoides. SUMMARY

[0005] In order to solve the problems existing in the current breeding of largemouth bass, and improve the breeding efficiency of largemouth bass, the application provides a SNP site combination for largemouth bass genotyping and an application thereof.

[0006] According to a first aspect of the application, a SNP site combination for largemouth bass genotyping is provided, which comprises 50000 SNP sites, and the positions and base information of the 50000 SNP sites on the chromosome are shown in Table 3.

[0007] The inventors of the present application collected fin tissues from 5 different largemouth bass population samples, extracted genomic DNA from the fin tissues and resequenced them, according to the resequencing results, individuals with a genetic relationship <0.25 were removed, then SNP molecular markers significantly associated with growth and development, sex determination, and resistance to largemouth bass iridovirus (LMBV) disease of largemouth bass were identified by GWAS analysis as a functional marker set (i.e. functional sites), then the functional marker set was filtered according to the SNP site loss rate, Hardy-Weinberg equilibrium test, minimum allele frequency, genotype loss rate, site depth annotation information and uniformity, and a background marker set (i.e. background sites) was obtained, finally, the functional marker set and the background marker set were integrated and de-redundant, thereby screening the 50000 SNP sites shown in Table 3, i.e. the SNP site combination for largemouth bass genotyping provided by the application. The SNP site combination for largemouth bass genotyping provided by the application is applied to the preparation of a largemouth bass whole genome liquid chip, which can quickly and accurately detect the genotype of a sample to be detected, the average detection rate of the SNP sites is as high as 99.95%, and the average consistency rate of the sample genotypes is as high as 99.95%, and the largemouth bass whole genome liquid chip has the characteristics of high capture specificity, low cost, flexible sites, etc., and is suitable for trait selection and improvement, gene mining, genetic relationship identification, genetic diversity analysis, etc. of largemouth bass, and can effectively improve the efficiency and precision of the selection of largemouth bass breeds, and is conducive to the cultivation of new varieties of largemouth bass that are more suitable for market demand and more competitive.

[0008] Preferably, the SNP site combination is screened by the following steps:

[0009] S1. Obtain samples of different largemouth bass populations, perform whole genome resequencing, and obtain a plurality of SNP sites according to the results of the whole genome resequencing;

[0010] S2. Screen the plurality of SNP sites obtained to obtain a SNP site combination for largemouth bass genotyping.

[0011] Preferably, in S1, the group of largemouth bass includes at least two of GAD 1, GAD 3, TAI, TUS, and WU.

[0012] Preferably, in S2, the screening of the obtained several SNP sites includes the following operation: obtaining the SNP data set after filtering the samples according to the kinship test ≥ 0.25 and the missing rate of the samples ≥ 0.2.

[0013] After obtaining several SNP sites by whole genome sequencing of different largemouth bass population samples, the individuals with kinship < 0.25 and missing rate < 0.2 are removed, and the samples with kinship test ≥ 0.25 and missing rate ≥ 0.2 are filtered to obtain the SNP data set for subsequent SNP site screening, which can further improve the specificity and accuracy of the SNP site combination for largemouth bass genotyping obtained by subsequent screening in the detection of largemouth bass genotyping.

[0014] Preferably, in S2, the screening of the obtained several SNP sites further includes the following operation: filtering the SNP sites meeting one of principles a, b, c, and d from the SNP data set.

[0015] a. Hardy-Weinberg equilibrium test ≤ 0.000001;

[0016] b. Minimum allele frequency MAF ≤ 0.05;

[0017] c. Genotype missing rate ≥ 0.1;

[0018] d. Site depth ≤ 1 / 2 of the average depth.

[0019] By filtering the SNP sites meeting one of the above principles a, b, c, and d, the specificity and accuracy of the SNP site combination for largemouth bass genotyping obtained by subsequent screening in the detection of largemouth bass genotyping can be improved to a certain extent.

[0020] According to a second aspect of the present application, a probe combination for detecting the above-mentioned SNP site combination for largemouth bass genotyping is provided.

[0021] According to a third aspect of the present application, the above-mentioned SNP site combination for largemouth bass genotyping is applied in the preparation of a largemouth bass genotyping liquid chip.

[0022] According to a fourth aspect of the present application, a largemouth bass whole genome liquid chip is provided, which includes a probe combination for identifying the genotype of the above-mentioned SNP site combination for largemouth bass genotyping.

[0023] According to a fifth aspect of the present application, there is provided an application of the above-mentioned whole-genome microarray of Micropterus salmoides in genotyping detection, genetic resource protection, kinship identification, whole-genome selection breeding, whole-genome association analysis, genetic diversity evaluation, and germplasm identification.

[0024] The SNP site group for genotyping Micropterus salmoides provided by the present application is applied in the preparation of the whole-genome microarray of Micropterus salmoides, which can be used for genotyping detection, genetic resource protection, kinship identification, whole-genome selection breeding, whole-genome association analysis, genetic diversity evaluation, and germplasm identification of Micropterus salmoides, and provides an important tool for molecular design breeding of Micropterus salmoides and plays an important role in the breeding research of Micropterus salmoides.

[0025] According to a sixth aspect of the present application, there is provided a detection method for genotyping Micropterus salmoides, comprising the following steps:

[0026] S1. obtaining genomic DNA of a Micropterus salmoides sample to be detected;

[0027] S2. detecting the above-mentioned genomic DNA by using the above-mentioned whole-genome microarray of Micropterus salmoides to obtain raw data;

[0028] S3. performing data analysis on the raw data to obtain the genotyping result of Micropterus salmoides. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The number distribution diagram of SNP sites in the Micropterus salmoides 50K whole-genome microarray provided in Example 2 on each chromosome.

[0030] Figure 2 The density distribution diagram of SNP sites in the Micropterus salmoides 50K whole-genome microarray provided in Example 2 on each chromosome.

[0031] Figure 3 The MAF distribution statistical result diagram of SNP sites in the Micropterus salmoides 50K whole-genome microarray provided in Example 2 on each chromosome.

[0032] Figure 4 The result diagram of PCA analysis of 322 different Micropterus salmoides samples by using 1373538 SNP sites obtained based on resequencing and 50000 SNP sites in the chip respectively provided in Example 4.

[0033] Figure 5 The SNP site detection rate statistical result diagram of 48 Micropterus salmoides samples detected by using the Micropterus salmoides 50K whole-genome microarray provided in Example 5.

[0034] Figure 6 The phylogenetic tree result map is constructed by using the genotyping results of 48 samples of Micropterus salmoides obtained in Example 5 and the chip sequencing genotyping results of 15 samples of Micropterus salmoides randomly selected in Example 6.

[0035] Figure 7 The whole genome association analysis result of the body length trait of Micropterus salmoides provided in Example 8.

[0036] Figure 8 The whole genome association analysis result of the body length trait of Micropterus salmoides provided in Example 8.

[0037] Figure 9 The whole genome association analysis result of the body length trait of Micropterus salmoides provided in Example 8.

[0038] Figure 10 The whole genome association analysis result of the body length trait of Micropterus salmoides provided in Example 8. DETAILED DESCRIPTION

[0039] The technical features in the technical solutions provided by the present application will be further clearly and completely described in combination with the specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person skilled in the art without creative labor fall within the protection scope of the present application.

[0040] Example 1 SNP site combination for genotyping of Micropterus salmoides

[0041] The present example provides a SNP site combination for genotyping of Micropterus salmoides, which is screened by the following steps:

[0042] 1. Selection of samples of different Micropterus salmoides populations

[0043] Five Micropterus salmoides populations (Gadefeng No. 1, Youlu No. 3, Tailu, Tulu and Wanlu) were collected, and the number of individuals selected from each population is shown in Table 1. The fin tissue samples collected from different Micropterus salmoides populations were placed in anhydrous ethanol for DNA extraction.

[0044] Table 1 Number of individuals of different Micropterus salmoides populations

[0045] Micropterus salmoides populations Gadwall 1 Super Blue 3 Taiwanese Blue Largemouth Bass Wan Bass Number 237 fish 28 fish 6 fish 22 fish 29 fish

[0046] 2. Sample sequencing and data quality control

[0047] (1) Genomic DNA extraction and quality control

[0048] Genomic DNA (gDNA) was extracted from the obtained fin tissue samples using a magnetic bead method. First, the integrity of the gDNA was tested using a 1.5% agarose gel. Then, the absorbance of the gDNA at 260 nm (A260) and 280 nm (A280) was measured using a NanoDrop 2000 nucleic acid and protein analyzer. The purity of the gDNA was determined by the A260 / A280 ratio. Finally, the gDNA in the sample was precisely quantified using a double-stranded DNA fluorescence quantitative detection kit (dsDNA HS Assay Kit for Qubit).

[0049] (2) Sequencing library construction and sequencing

[0050] ①Use DNA Universal Library Construction Kit (for MGI) is used to construct sequencing libraries. Genomic DNA is then processed using Covaris. TM Fragments of approximately 300–350 bp in length were randomly fragmented, end-repaired, A-tailed, and ligated with sequencing adapters. SP Beads are used to screen DNA samples with a length of approximately 300–350 bp, which are then amplified by PCR and reused. SP Beads were used to purify PCR products, and finally, sequencing libraries were obtained.

[0051] ② After the sequencing library is constructed, first use Preliminary quantification was performed using a 2.0 fluorometer, followed by... (Agilent) checks the inserted fragments in the library, and if they meet the expectations, they are then sequenced.

[0052] ③ After the library passes quality control, pooling is performed based on the effective concentration of the library and the required amount of data for sequencing. The DNBSEQ-T7 sequencer is used, and the sequencing strategy of PE150 is selected for sequencing. The raw image data obtained from sequencing is converted into raw sequence data (raw reads) by base calling software and stored in FASTQ file format.

[0053] (3) Sequencing data quality control and SNP detection

[0054]

[0055] (2) Clean Reads were aligned to the reference genome (GenBank accession number: ASM1485139v1 [GCF_014851395.1, Female] and CAFS_Msal_1 [GCA_036785525.1, male]) by using BWA 0.7.17 software to obtain a Bam file, then sorted and indexed using Samtools 1.7 tools, and then using the module provided by GATK 4.1.8.0 software to remove duplicates, and then using GATK 4.1.8.0 software to screen and detect SNP sites, and then using the VariantFiltration module to strictly filter the SNP sites, and using the Annovar software to annotate the obtained SNP sites. The SNP site filtering parameters are as follows: QD < 2.0, FS > 60.0, MQ < 40.0, SOR > 3.0, MQRankSum < -12.5, and ReadPosRankSum < -8.0.

[0056] (4) SNP site quality control

[0057] From the SNP sites obtained above, the SNP data set was obtained by filtering the samples according to the kinship test ≥ 0.25, the sample deletion rate ≥ 0.2, and the PCA result (outlier sample). Then, the SNP sites meeting the following indicators were deleted: Hardy-Weinberg equilibrium test ≤ 0.000001, minimum allele frequency MAF ≤ 0.05, genotype deletion rate ≥ 0.1, and site depth ≤ 1 / 2 of the average depth. Finally, 322 samples were retained, and 1373538 SNP sites were obtained.

[0058] (5) SNP site screening

[0059] Table 2 SNP sites significantly associated with growth, gender, and resistance to largemouth bass iridovirus (LMBV) disease and other traits

[0060] ​

[0061]

[0062] Note: In Table 2, ref represents the reference base (also referred to as the reference allele), and alt represents the mutant base (also referred to as the alternative allele); the 23 chromosomes of the reference genome (Genome assembly) ASM1485139v1 (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_014851395.1 / ) of Micropterus salmoides are assembled at the chromosome level, and the corresponding chromosome names are Ms_hic_scaffold_1-23, and Ms_hic_scaffold_1, 2, 3, 5, 6, 8, 13, 17, 18, 20 in Table 2 represent the first, second, third, fifth, sixth, eighth, thirteenth, seventeenth, eighteenth, and twentieth chromosomes, respectively, in the reference genome ASM1485139v1 assembled at the chromosome level.

[0063] According to the principle of uniform distribution, SNP sites are screened on the whole genome, and a sliding window method is used, i.e., a window is slid on the genome with 10Kb as a window and 10Kb as a step, and according to the variation type of SNP annotation, the relevant SNP sites are retained in the order of stopgain = stoploss > splicing > nonsynonymous SNP, and if there is no SNP site with the above-mentioned variation types in the window, SNP sites are selected according to low deletion rate and high MAF, to obtain SNP site set 1; at the same time, 45 SNP sites (Table 2) significantly associated with growth, sex, and resistance to LMBV (Largemouth bass virus) disease and other traits obtained in the previous genome-wide association study (GWAS) are added to SNP site set 1 to form a new SNP site set 2; SNP site set 2 is evaluated to determine whether the site can be designed for a probe, and the specificity, complexity, and GC content of the sequence upstream and downstream of the target site are mainly evaluated, and the target site is preferentially placed in the middle position of the probe, and the designed probe is 120bp in length; through strict screening, 50,000 SNP sites are finally obtained, i.e., the SNP site combination provided in the present embodiment for genotyping of Micropterus salmoides, and the positions and base information of the 50,000 SNP sites on the chromosomes are shown in Table 3.

[0064] Table 3 SNP site combination for genotyping of Micropterus salmoides

[0065]

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[0080]

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[0084]

[0085]

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[0090]

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[0110]

[0111]

[0112] Note: In Table 3, the 23 chromosomes of the chromosome level of the reference genome (Genome assembly) ASM1485139vl (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_014851395.1 / ) of Micropterus salmoides are assembled, and the corresponding chromosome names are Ms_hic_scaffold_1~23, respectively. Ms_hic_scaffold_1~23 in Table 3 represent the first~23th chromosome in the corresponding chromosome level of the reference genome ASM1485139vl of Micropterus salmoides, respectively.

[0113] Example 2 Micropterus salmoides 50K Whole Genome Liquid Chip

[0114] The present example provides a Micropterus salmoides 50K whole genome liquid chip, which comprises a probe combination for identifying the genotypes of the SNP site combination for Micropterus salmoides genotyping screened in Example 1.

[0115] The 50,000 SNP sites (SNP site combination for Micropterus salmoides genotyping) screened in Example 1 are applied to probe design, and the design principle of the probe is as follows: the 60-80 bp genomic sequence before and after each target site is used as the probe design sequence of the SNP molecular marker, the probe sequence length is 120 bp, the GC content is between 20-80%, the Tm value is 60-80℃, the number of alignments on the reference genome is ≤5 (to evaluate the probe design effect), and each SNP site has at least one probe covering. m

[0116] Based on the above probe design principle, the liquid phase capture probes are synthesized by Beijing ComforSciences Agricultural Technology Co., Ltd., a total of 49,997 capture probes are synthesized, the CAGT targeted capture sequencing technology is used to complete the genotyping of the target SNP site, the CAGT targeted capture sequencing technology first constructs a whole genome resequencing library for the sample to be tested, then according to the DNA complementary principle, the biotin-modified probe is hybridized with the target region of the genome to form a double-stranded structure, then the streptavidin-coated magnetic beads are used to adsorb the probe carrying biotin modification, thereby capturing the library structure containing the target SNP site, finally, the captured target library sequence is eluted, amplified and sequenced, and finally the genotype of the target SNP site is obtained. According to the probe design results, the probes that cannot be uniquely aligned on the genome and contain repeat sequences in the flanking sequence are removed, and the designed probes are applied to the preparation of the liquid chip, and finally a Micropterus salmoides 50K whole genome liquid chip is obtained. The number of SNP sites in each chromosome in the chip is shown in Figure 1 , the density distribution of SNP sites in each chromosome is shown in Figure 2 , and the minor allele frequency (MAF) distribution of SNP sites in each chromosome is shown in Figure 3 .

[0117] As can be seen from Figure 1 and Figure 2 , the SNP sites in the Micropterus salmoides 50K whole genome liquid chip are mainly concentrated on the Ms_hic_scaffold_1-23 chromosomes of Micropterus salmoides, and the SNP sites in the Micropterus salmoides 50K whole genome liquid chip are uniformly distributed on the chromosomes; as can be seen from Figure 3 ​It can be seen that the SNP sites with MAF of 0.4-0.5 are the most, indicating that the SNP site diversity of the 50K whole genome liquid chip of Micropterus salmoides is high.

[0118] Embodiment 3

[0119] A detection method for genotyping of Micropterus salmoides, comprising the following steps:

[0120] S1. Obtain the genomic DNA of the Micropterus salmoides sample to be detected;

[0121] S2. Detect the genomic DNA by using the 50K whole genome liquid chip of Micropterus salmoides provided in Embodiment 2 to obtain raw data;

[0122] S3. Perform data analysis on the raw data to obtain the genotyping result of the Micropterus salmoides.

[0123] Embodiment 4

[0124] This embodiment aims to use the 1,373,538 SNP sites initially obtained by resequencing in Embodiment 1 and the 50,000 SNP sites in the chip to respectively perform principal component analysis (PCA analysis) on 322 different Micropterus salmoides samples, and the analysis results are shown in Figure 4 , wherein, Figure 4 A is an analysis result graph obtained by plotting according to the SNP sites of the sample resequencing, Figure 4 B is an analysis result graph obtained by plotting according to the detection of the SNP sites of the sample by using the 50K whole genome liquid chip of Micropterus salmoides provided in Embodiment 2.

[0125] From the PCA analysis results of Figure 4 , it can be seen that the sample clustering of the resequencing SNP sites and the SNP sites of the sample detected according to the 50K whole genome liquid chip of Micropterus salmoides are consistent, indicating that the 50K whole genome liquid chip of Micropterus salmoides provided in Embodiment 2 has good representativeness and can be well applied to the genotyping detection of Micropterus salmoides.

[0126] Embodiment 5 Effect evaluation of the 50K whole genome liquid chip of Micropterus salmoides in the genotyping detection of Micropterus salmoides samples

[0127] The present example aims to use the 50K whole genome liquid chip of largemouth bass provided in Example 2 to detect largemouth bass samples to evaluate the effect of the 50K whole genome liquid chip of largemouth bass in the typing detection of largemouth bass samples, so as to verify the typing quality of the 50K whole genome liquid chip of largemouth bass. The specific operation steps are as follows: 48 fish fin tissues of largemouth bass (including 3 pairs of technical repeat samples) are collected as test samples, DNA extraction, library construction is carried out, and the 50K whole genome liquid chip of largemouth bass provided in Example 2 is used to enrich the DNA fragments in the chip target site region by probe hybridization capture, and the barcode sequence is added to the DNA of each sample to be tested. Sequencing is carried out by using the principle of second-generation sequencing, the target site and its flanking sequence information are obtained, and the SNP site typing is carried out by using the method involved in Example 1 and the method provided in Example 3. The results are shown in Table 4 and Table 5. Figure 5 At the same time, the typing results of 3 pairs of technical repeat samples (LS4 and LS4r, TL2 and TL2r, TL3 and TL3r) are analyzed, and the results are shown in Table 5.

[0128] Table 4 SNP site typing results of 48 largemouth bass samples

[0129]

[0130]

[0131] Table 5 SNP site typing result analysis of 3 pairs of technical repeat samples

[0132] Test sample number Number of SNP sites with consistent typing Number of SNP sites in common Consistency rate of typing TL2 and TL2r 49827 49852 99.95％ TL3 and TL3r 49823 49838 99.97％ LS4 and LS4r 49887 49922 99.93％

[0133] As shown in Table 4 and Table 5, Figure 5 It can be seen that the 50K whole genome liquid chip of largemouth bass provided in Example 2 is used for SNP site typing detection of 48 largemouth bass samples, and the average SNP site detection rate of each sample is as high as 99.95%.

[0134] As shown in Table 5, during the SNP site typing detection of 48 largemouth bass samples by using the 50K whole genome liquid chip of largemouth bass provided in Example 2, the typing consistency of the 3 pairs of technical repeat samples involved is as high as 99.95%.

[0135] Example 6 typing detection

[0136] This example aims to detect the sample of Micropterus salmoides by using the 50K whole genome liquid phase chip of Micropterus salmoides provided in Example 2, and the specific operation steps are as follows: 136 fish fin tissues of Micropterus salmoides are collected as test samples, DNA extraction, library construction are carried out, and the DNA fragments in the chip target site region are enriched by probe hybridization capture by using the 50K whole genome liquid phase chip of Micropterus salmoides provided in Example 2, and the barcode sequence is added to the DNA of each sample to be tested, and the principle of second-generation sequencing is used for sequencing, after obtaining the target site and its flanking sequence information, the method involved in Example 1 and the method provided in Example 3 are used for SNP site typing, and the chip sequencing typing results of 15 Micropterus salmoides samples randomly selected from the SNP site typing results of the above 136 Micropterus salmoides samples are used for subsequent experiments.

[0137] Example 7 Application of 50K Whole Genome Liquid Phase Chip of Micropterus salmoides in Population Structure Analysis of Micropterus salmoides

[0138] This example aims to use the typing results of 48 Micropterus salmoides samples obtained in Example 5 and the chip sequencing typing results of 15 Micropterus salmoides samples (ZJ11, ZJ12, ZJ14, ZJ15, ZJ18, ZJ28, ZJ31, ZJ54, ZJ55, ZJ64, ZJ70, ZJ87, ZJ92, ZJ107, ZJ122) randomly selected in Example 6, to infer the phylogenetic relationship by using Phylip software, and after data conversion by using Mega software, to construct an evolutionary tree by using the neighbor joining method (neighbor joining method) therein, and the result is shown in Figure 6

[0139] As can be seen from Figure 6 , the 50K whole genome liquid phase chip of Micropterus salmoides obtained in Example 2 can effectively distinguish Micropterus salmoides from different sources, and can reflect their phylogenetic relationship.

[0140] Example 8 Whole Genome Association Analysis of Important Economic Traits Based on 50K Whole Genome Liquid Phase Chip of Micropterus salmoides

[0141] ​The embodiment aims to use the 50K whole genome liquid chip of Micropterus salmoides provided in embodiment 2 to perform whole genome association analysis (GWAS) on the growth traits of full length, body length, body height and body weight of Micropterus salmoides. The specific operation steps of SNP site typing refer to embodiment 6. The SNP sites of 136 Micropterus salmoides samples are screened according to the missing rate ≤0.1 and MAF ≥0.05 by Plink software (--geno 0.1 --maf 0.05), 48587 SNP sites of 136 data are used for GWAS analysis, rMVP software is used to perform whole genome association analysis on the four growth traits of full length, body length, body height and body weight, three calculation models of general linear model (GLM), mixed linear model (MLM) and FarmCPU model are used, and the principal component is used as a covariate for correction. The whole genome association analysis results of body length are shown in Figure 7 , the whole genome association analysis results of full length are shown in Figure 8 , the whole genome association analysis results of body weight are shown in Figure 9 , and the whole genome association analysis results of body height are shown in Figure 10 .

[0142] From the whole genome association analysis results of the growth traits shown in Figure 7 , 8 , 9, 10, the SNP sites significantly associated with the four growth traits of full length, body length, body height and body weight are found. The 10th chromosome (Ms_hic_scaffold_10) and the 16th chromosome (Ms_hic_scaffold_16) corresponding to the chromosome level of the reference genome ASM1485139v1 assembled of Micropterus salmoides are the main chromosomes related to the growth traits of Micropterus salmoides. Therefore, the whole genome association analysis of the growth traits of Micropterus salmoides using the 50K whole genome liquid chip of Micropterus salmoides provided by the present application can obtain relatively accurate whole genome association analysis results.

[0143] In summary, the present inventors screened 50000 SNP sites shown in Table 3 from 5 different Micropterus salmoides population samples, which are the SNP site combinations provided by the present application for genotyping of Micropterus salmoides. The SNP site combinations provided by the present application for genotyping of Micropterus salmoides are applied to the preparation of Micropterus salmoides whole genome liquid chip, which can be used for genotyping detection, genetic resource protection, kinship identification, whole genome selection breeding, whole genome association analysis, genetic diversity assessment, germplasm identification and the like of Micropterus salmoides, providing an important tool for molecular design breeding of Micropterus salmoides, and playing an important role in the breeding research of Micropterus salmoides.

[0144] The above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application is described in detail with reference to the above examples, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently, and these modifications or replacements are within the protection scope of the present application.

Claims

1. A probe combination for detecting a combination of SNP loci for genotyping of largemouth bass, characterized by: The SNP site combination for largemouth bass genotyping consists of 50000 SNP sites, and the reference genome of the 50000 SNP sites is ASM1485139v1; The position and base information of the 50000 SNP sites on the chromosome are as follows:

2. A Micropterus salmoides whole genome liquid chip, characterized in that: The largemouth bass whole genome liquid chip includes the probe combination for detecting the SNP site combination for largemouth bass genotyping according to claim 1.

3. The application of the largemouth bass whole genome liquid chip according to claim 2 in largemouth bass genotyping detection, genetic resource protection, kinship identification, whole genome selection breeding, whole genome association analysis, genetic diversity evaluation, and germplasm identification.

4. A detection method for genotyping of largemouth bass, characterized by, Comprising the following steps: S1. Obtaining the genomic DNA of the largemouth bass sample to be tested; S2. Detecting the genomic DNA using the largemouth bass whole genome liquid chip according to claim 2 to obtain raw data; S3. Analyzing the raw data to obtain the genotyping result of the largemouth bass.

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