Micropterus salmoides SNP chip and application thereof

Abstract

The invention belongs to the technical field of fish breeding and biochips, and particularly relates to a micropterus salmoides SNP (Single Nucleotide Polymorphism) chip and application thereof. A specific capture probe is designed based on the provided largemouth bass SNP molecular marker combination, a gene chip for whole genome genotyping of largemouth bass is constructed, the gene chip is used for accurate and comprehensive detection of whole genome genotyping of largemouth bass, and the gene chip has the advantages of high detection flux, low cost, flexible customization and the like; the method is used for germplasm resource identification, genetic diversity analysis, whole genome association analysis, important functional gene mining and selective breeding of the micropterus salmoides, and a novel and efficient tool is provided for accelerating fine variety breeding of the micropterus salmoides.

Description

Technical Field

[0001] This invention belongs to the field of fish breeding and biochip technology, specifically relating to a largemouth bass SNP chip and its application. Background Technology

[0002] Largemouth bass are an important freshwater aquaculture species in my country. With the expansion of the industry, breeding varieties with fast growth rates and strong resistance (such as heat tolerance) has become a core requirement for the industry's development.

[0003] Traditional breeding methods mainly rely on phenotypic selection, which has drawbacks such as long breeding cycles and low efficiency in selecting recessive traits and traits that are difficult to measure in vivo. Genomic selection (GS), as an advanced molecular breeding method, can significantly improve the accuracy of breeding value estimation.

[0004] Currently, genotyping of largemouth bass mainly relies on whole-genome resequencing. Resequencing is costly, has fixed loci that cannot be flexibly adjusted, and contains a large amount of redundant information. Liquid-phase microarrays, based on targeted capture sequencing technology, offer advantages such as low cost, flexible customization, uniform locus distribution, and high accuracy. However, there is currently a lack of medium- to low-density liquid-phase breeding microarrays designed for a balanced development of growth and heat tolerance traits in largemouth bass, while also possessing high predictive accuracy. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a genotyping solution for the largemouth bass genome that features scientifically designed and precise loci, high detection throughput, low cost, and is specifically targeted at the core economic traits of the largemouth bass. This solution can be applied to important trait selection, pedigree identification, and genetic diversity management in largemouth bass. The chip constructed based on this genotyping solution exhibits high capture rate, high genotyping accuracy, and low detection cost, making it a highly efficient genotyping tool suitable for genomic selection breeding and basic research in largemouth bass.

[0006] This invention provides a largemouth bass SNP molecular marker combinatorial, comprising 6112 SNP molecular markers, the information of which on the largemouth bass genome version GCA_224357851 is shown in Table 1 of the specification.

[0007] This invention provides a method for screening SNP molecular marker combinations in largemouth bass, comprising the following steps: (1) Whole genome resequencing was performed on all largemouth bass samples that underwent heat stress and pathogen infection experiments to obtain whole genome sequence information, and phenotypic data of heat tolerance, disease resistance, morphological traits and growth traits of all largemouth bass samples were statistically analyzed. (2) The whole genome sequence information was compared with the largemouth bass genome version GCA_224357851. After quality control filtering, SNP molecular markers were extracted to obtain whole genome resequencing data. The quality control filtration includes: 1) First, filter according to QD<2.0, QUAL<30.0, MQ<40.0, FS>60.0, SOR>3.0, MQRankSum<-12.5, and ReadPosRankSum<-8.0; 2) Then, based on a missing rate of <20% and Hardy-Weinberg equilibrium >10... -6 SNPs with a minimum allele frequency >0.05 were retained, and missing SNPs were filled in. (3) Perform whole-genome association analysis on the whole-genome resequencing data and the phenotypic data of heat resistance, disease resistance, morphological traits and growth traits, distinguish the core SNP molecular markers and background SNP molecular markers related to the phenotypic data, and after quality control filtering of background SNP molecular markers, obtain at least 6112 SNP molecular markers based on the fact that the background SNP molecular markers uniformly cover the whole genome and that there are no duplicate core SNP molecular markers within 50kb upstream and downstream of the background SNP molecular markers, as shown in Table 1 of the specification, including 4821 background SNP molecular markers, 435 heat resistance-related SNP molecular markers, 436 disease resistance-related SNP molecular markers, and 420 morphological and growth trait-related SNP molecular markers; The background SNP molecular markers were subjected to a more stringent standard than the quality control filtering (initial filtering) in step (2), namely, requiring the minimum allele frequency of these loci to be >0.1 and the deletion rate to be <10%.

[0008] The SNPs screened in this invention that are related to heat resistance (high temperature resistance) are located in heat shock protein family genes, energy metabolism pathway genes, and oxidative stress response genes; the SNPs related to disease resistance are located in major histocompatibility complex genes, Toll-like receptor genes, and immunoglobulin-like genes; and the SNPs related to morphological and growth traits are located in growth hormone genes, insulin-like growth factor genes, and myostatin genes.

[0009] Based on the design principles of 110bp length, GC content of 30%-70%, and maximum number of similar fragments of 5, molecular probes with 6112 SNP molecular markers as shown in Table 1 of the specific targeting specification were designed.

[0010] This invention provides an SNP molecular probe set, wherein the SNP molecular probes contained in the SNP molecular probe set target at least the 6112 largemouth bass SNP molecular markers shown in Table 1 of the specification. Further, the SNP molecular probes contained in the SNP molecular probe set have a length of 110 bp and a GC content between 30% and 70%, specifically binding to the 6112 largemouth bass SNP molecular markers shown in Table 1 of the specification.

[0011] Gene chips were constructed using molecular probes containing the 6112 SNP molecular markers shown in Table 1 of the specific targeting instructions, resulting in a whole-genome SNP chip for the largemouth bass. Targeted capture sequencing technology was employed, offering advantages such as high throughput, low cost, and flexible customization.

[0012] The present invention relates to the application of a SNP molecular probe set provided above in the preparation of a whole genome SNP chip product for largemouth bass.

[0013] This invention provides a whole-genome SNP chip for largemouth bass, containing a set of SNP molecular probes provided above. The whole-genome SNP chip for largemouth bass is a liquid-phase chip.

[0014] The present invention provides a kit containing an SNP molecular probe set provided above and / or a largemouth bass whole genome SNP chip provided above.

[0015] The above-mentioned largemouth bass whole genome SNP chip and / or the above-mentioned kit provided by the present invention are used for germplasm resource identification, genetic diversity analysis, genome-wide association analysis, important functional gene mining and selection breeding of largemouth bass. The selection breeding includes variety identification, parentage identification, population identification, pedigree reconstruction, inbreeding level assessment, optimized mating scheme, fixation of superior genes, trait assessment and prospect selection, etc. The associated traits of the trait assessment include at least one of heat tolerance, disease resistance, morphological traits and growth traits. The morphological traits are selected from at least one of body width, body height, body length, head length, head height, caudal peduncle height, caudal peduncle length, fork length, etc. The growth traits are selected from at least one of body weight, body weight gain rate, body length gain, specific growth rate, condition factor, etc.

[0016] This invention provides a method for obtaining the whole genome genotype of largemouth bass, comprising the following steps: (1) Extract genomic DNA from the largemouth bass samples and construct sequencing libraries; (2) Use the whole genome SNP chip of the largemouth bass provided above and / or the kit provided above to perform hybridization capture to obtain the captured fragment; (3) High-throughput sequencing was performed using the captured fragments and compared with the reference genome of largemouth bass (GCA_224357851 version) to obtain the genotyping data of the whole genome of largemouth bass.

[0017] This invention provides a method for evaluating the breeding of largemouth bass, comprising the following steps: obtaining genotyping data of largemouth bass statistical samples based on the method for obtaining largemouth bass whole genome genotyping provided above; constructing a largemouth bass breeding value prediction model using a genomic breeding value statistical model; inputting the genotyping data of the largemouth bass sample to be tested based on the method for obtaining largemouth bass whole genome genotyping provided above; obtaining the breeding value of the largemouth bass sample to be tested; and conducting a breeding evaluation of largemouth bass.

[0018] The statistical model for the genomic breeding value is selected from any one of GBLUP (Genome Best Linear Unbiased Prediction), ABLUP (Pedagogical-Based BLUP), SSGBLUP (One-Step GBLUP for Single and Multiple Traits), CNN (Convolutional Neural Network), and MGIDI (Multi-Track Selection Index), with GBLUP being preferred. Further, a GBLUP prediction model is constructed using the GBLUP model to connect genotyping data with phenotypic traits. The genotyping data of the largemouth bass sample to be tested is input based on the largemouth bass whole-genome genotyping method provided above in this invention to obtain the breeding value of the largemouth bass sample for breeding evaluation.

[0019] The phenotypic traits include heat resistance, disease resistance, morphological traits and growth traits. The morphological traits are selected from at least one of body width, body height, body length, head length, head height, caudal peduncle height, caudal peduncle length, fork length, etc. The growth traits are selected from at least one of body weight, body weight gain rate, body length gain, specific growth rate, conditionability, etc.

[0020] This invention utilizes largemouth bass SNP molecular markers, probes targeting these markers, and a chip constructed from them. It pioneers a site combination model of "precisely selected core sites (or foreground sites) + globally assessed (covering the entire genome) background sites." The core sites comprehensively consider site polymorphism, genome distribution evenness, and association with key breeding traits, enabling rapid and precise selection of genes for key traits (efficacy), significantly improving the efficiency of improving key traits such as heat tolerance, disease resistance, and growth. Background sites ensure the genome coverage required for whole-genome selection, allowing for unbiased estimation of the overall breeding value of individuals. This approach is both universal and targeted, achieving "one chip, multiple uses," greatly improving efficiency and comprehensiveness, meeting the information requirements of polymorphism, and significantly enhancing breeding accuracy and efficiency, satisfying breeding precision requirements. Furthermore, compared to whole-genome resequencing, it simplifies the genome sequencing process and improves reproducibility.

[0021] The beneficial effects of this invention are as follows: 1. Balanced design of multiple traits: The chip of this invention not only covers growth-related traits, but also specifically enriches core sites related to heat resistance and disease resistance, which solves the problem of decreased resistance caused by the single pursuit of growth rate in traditional breeding, and achieves dual improvement of "growth-resistance".

[0022] 2. Excellent and stable predictive performance. After 10 repeated 5-fold cross-validation, the chip performed well in the genome prediction of the main morphological traits of largemouth bass, improving the stability of breeding value estimation.

[0023] 3. Low cost and high flexibility: Compared with traditional solid-phase chips, the liquid-phase chip detection cost of this invention is reduced by more than 40%. Furthermore, based on targeted capture technology, it can detect not only 6112 target SNP sites but also new variations in the surrounding areas of the sites, providing a large amount of information. The site library can also be upgraded at any time according to breeding needs.

[0024] 4. The genotype data obtained by the chip of this invention, combined with the GBLUP prediction model, can enable early and accurate evaluation of phenotypic breeding values ​​such as body weight, heat tolerance, and disease resistance of largemouth bass. This fills the gap in the independent research and development of low-density breeding chips for largemouth bass and is of great significance for accelerating the breeding process of superior varieties of largemouth bass. Attached Figure Description

[0025] Figure 1 This is a schematic diagram showing the physical distribution of the SNP sites of the present invention on the 23 chromosomes of the whole genome of the largemouth bass.

[0026] Figure 2 This is a statistical diagram showing the frequency distribution of physical distances between adjacent SNP sites in the liquid phase chip of this invention.

[0027] Figure 3 This is a statistical diagram showing the minimum allele frequency distribution of the SNP sites in this invention.

[0028] Figure 4 This diagram illustrates the distribution of the SNP sites of the present invention across different functional regions (such as intergenic regions, intronic regions, and exon regions) in the whole genome of the largemouth bass.

[0029] Figure 5 This is a survival curve of the heat-resistant GEBV population of largemouth bass, calculated based on the liquid-phase chip typing data of this invention, in an actual heat stress experiment.

[0030] Figure 6 This is a cross-validation accuracy comparison chart of seven traits of largemouth bass based on the liquid-phase chip genotyping data of this invention. The traits include body height, body length, body weight, caudal peduncle height, caudal peduncle length, head length, and body fat percentage.

[0031] Figure 7 This is a schematic diagram of principal component analysis (PCA) clustering of different populations of largemouth bass based on the liquid phase chip typing data of this invention. Detailed Implementation

[0032] Example 1: Screening of 6112 SNP loci in largemouth bass and preparation of liquid-phase breeding chips (1) Construction of the largemouth bass sample population Thirty-fivety-three largemouth bass were randomly selected for a rigorous heat stress experiment. Their survival time and weight changes at critical high temperatures were recorded to screen for heat tolerance and growth-related SNPs. An additional 101 individuals were selected for a pathogen infection experiment to evaluate their disease resistance and immune survival status, and to screen for disease resistance-related SNPs.

[0033] (2) Whole-genome resequencing NGS and variant detection DNA was extracted from fin tissue using a kit, and the extracted DNA samples were stored at -80°C for library construction. DNA concentration was determined using Nanodrop. Sequencing libraries were constructed using 1 µg of genomic DNA using the Hieff NGS® OnePot II DNA library preparation kit and high-throughput sequencing was performed on an Illumina Novaseq 6000 platform (Illumina, USA). GCA_224357851 was then used as the largemouth bass genome reference sequence. SNPs were identified using GATK and filtered according to the following criteria: QD < 2.0, QUAL < 30.0, MQ < 40.0, FS > 60.0, SOR > 3.0, MQRankSum < -12.5, and ReadPosRankSum < -8.0. Subsequently, PLINK software was used for quality filtering of SNP sites, retaining sites that met the following conditions: SNP deletion rate < 20%, Hardy-Weinberg equilibrium > 10. -6 The minimum allele frequency was >0.05. Missing SNPs were filled using Beagle v5.1, and the processed SNPs were used for subsequent analysis.

[0034] (3) Screening of core sites Using resequencing and phenotypic data from 454 individuals, genome-wide association analysis (GWAS) was performed using a mixed linear model (MLM) to screen for 435 SNPs associated with heat tolerance, 420 SNPs associated with growth, and 436 SNPs associated with disease resistance. Specifically, an algorithm was used to ensure that no duplicate core markers were present within a 50kb physical range upstream and downstream of any background locus, effectively reducing linkage disequilibrium redundancy and improving the computational efficiency and representativeness of the microarray. This invention further screened 4821 background loci according to the principle of uniform distribution, and applied more stringent standards to their quality than the initial filtering, requiring these loci to have a minimum allele frequency >0.1 and a deletion rate <10%; finally, 6112 SNP loci were selected as microarray loci. Figure 1 ).

[0035] (4) Liquid phase chip fabrication and performance testing For the 6112 selected loci, specific probes with a length of 110 bp and a GC content between 30% and 70% were designed to prepare a liquid-phase SNP chip for largemouth bass, named GenoBaits® Flame Bass No. 1 Largemouth Bass 5K Breeding Chip. Performance test results showed that the sample locus detection rate was between 99.64% and 100%, with an average detection rate of 99.92%. Furthermore, the SNP loci included in this chip exhibited good polymorphism in various natural populations of largemouth bass, making it suitable for genetic background analysis of breeding populations. Statistical analysis revealed that the average physical distance between loci was 134,711 bp; the maximum inter-locus distance across the entire genome was 843,824 bp, with no large-scale genetic map gaps. This distribution characteristic ensures that the chip can effectively capture linkage disequilibrium signals across the entire genome, guaranteeing the accuracy of breeding value estimation. Figure 2 99.29% of SNP loci have a minimum allele frequency >0.05 ( ). Figure 3 Of these, there are 1291 core SNPs, including 435 SNPs related to heat tolerance, 420 SNPs related to growth, 436 SNPs related to disease resistance, and 4821 background sites evenly covering the entire genome. In terms of location, there are 3451 SNPs located between genes and in regulatory regions, 2436 SNPs located in intron regions, and 225 SNPs located in exons. Figure 4 ).

[0036] Table 1 Location information of SNP sites Example 2: Accuracy Assessment and Practical Verification of Predictive Heat Tolerance Traits for Largemouth Bass This invention further evaluates the application effect of the liquid-phase SNP chip with 6112 SNP loci in precision breeding through actual prediction and phenotypic verification of the heat tolerance trait in largemouth bass. In the verification process, firstly, the genotype data and heat tolerance phenotype records of 353 reference population individuals obtained through second-generation whole-genome resequencing in Example 1 are integrated, and a genomic selection prediction scheme is constructed using a GBLUP statistical model. Subsequently, the liquid-phase SNP chip developed in this invention is used to efficiently genotype 336 individuals in a test population independent of the reference population, obtaining their whole-genome SNP locus information. By substituting the chip genotyping data of the test population into the constructed prediction model, the estimated genomic breeding value (GEBV) of heat tolerance for each individual in the test population is estimated.

[0037] To visually verify the biological reliability of the prediction results, this embodiment rigorously selected the top 30 individuals with high breeding value (estimated strong heat tolerance) and the bottom 30 individuals with low breeding value (estimated weak heat tolerance) from a test population of 336 individuals based on the GEBV ranking results, forming a high breeding value group and a low breeding value group, respectively. Subsequently, both groups were placed under a uniform extreme high-temperature heat stress environment for a challenge experiment, and the survival time of each individual was precisely recorded. The experimental statistical results clearly show that the average survival time of individuals in the high breeding value group was significantly better than that in the low breeding value group, and the difference between the two reached a highly significant level (P<0.01). This result is as follows... Figure 5 As shown.

[0038] Example 3: Five-fold cross-validation and heritability calculation of body size trait in largemouth bass This invention further utilizes a developed liquid-phase SNP chip to verify the accuracy of genetic parameter estimation and genome prediction for seven important body shape traits in 1994 largemouth bass. During the experiment, the whole-genome SNP genotype information of the experimental population was first obtained using the chip, and combined with the measured phenotypic data, a genome phylogenetic matrix was constructed using GCTA software. Subsequently, for seven traits—body height, body length, body weight, caudal peduncle height, caudal peduncle length, head length, and condition factor—variance component estimation was performed using restricted maximum likelihood (REML).

[0039] Table 2. Heritability and Standard Error of 7 Traits The experimental statistical results are shown in Table 2. The chip of this invention can accurately capture the genetic variation of each trait. Calculations showed that the additive genetic variance, environmental variance, and phenotypic variance of these seven physical traits all exhibited good statistical characteristics, and the standard errors (SE) of each parameter were all within a low and controllable range. The results showed that the narrow-sense heritability of the seven traits ranged from 0.12 to 0.29, belonging to the low to medium level of heritability. Among them, body weight had the lowest heritability (0.12), while body fat had the highest (0.29).

[0040] Based on the assessment of genetic parameters, this invention further validated the genome prediction capability of the aforementioned seven phenotypic traits using the GBLUP model. Through 10 replicates of 5-fold cross-validation, the prediction accuracy was calculated by dividing the correlation coefficient between the predicted breeding value and the observed phenotypic value by the square root of the trait heritability. The results showed that the chip exhibited very robust prediction accuracy for the seven traits, with all prediction accuracies exceeding 0.5. The specific trends are as follows: Figure 6 As shown in the figure. This result fully demonstrates that the combination of 6112 SNP sites developed in this invention can achieve high-precision prediction of complex body shape traits in largemouth bass by capturing linkage disequilibrium information across the entire genome, providing solid data support for molecular-assisted breeding and precise improvement of largemouth bass body shape traits.

[0041] Example 4: Population verification of largemouth bass This invention further validates the robustness and discriminative ability of the liquid-phase microarray with 6112 SNP loci under different genetic backgrounds through genetic cluster analysis of large-scale samples from multiple populations. This embodiment utilizes PLINK software to integrate all largemouth bass experimental populations involved in Examples 1, 2, and 3, totaling 2683 individuals. By extracting genotyping data from the core loci of these individuals, principal component analysis (PCA) is performed to evaluate the recognition efficacy of this microarray locus combination for different populations.

[0042] Analysis results as follows Figure 7 As shown, the genetic component information obtained based on this chip can clearly divide the reference population, test population, and validation population into three independent genetic clusters in two-dimensional space, with distinct boundaries and no significant overlap between the populations. This remarkable population stratification effect fully demonstrates that the SNP locus combination selected in this invention has extremely high population sensitivity and genetic representativeness, not only accurately capturing subtle variations within a single breeding line but also effectively identifying genetic differences between individuals from different sources.

[0043] Finally, it should be noted that the above results fully demonstrate that the 6112 core SNP loci obtained by screening based on this invention and the liquid phase SNP chip constructed for genome selection breeding of largemouth bass can provide high-throughput genotyping data of the whole genome quickly and accurately, providing core data support for genetic analysis, germplasm identification and whole genome selection of the target population.

[0044] The embodiments described above are merely illustrative of the technical solutions and preferred embodiments of the present invention, and are not intended to limit the scope of protection of the present 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 without departing from the spirit of the present invention. All modifications, improvements, or equivalent substitutions made to the technical solutions of the present invention within the spirit and principles of the present invention shall not cause the essence of the corresponding technical solutions to depart from the spirit and scope of the technical solutions of the embodiments of the present invention, and shall all fall within the scope of protection defined by the claims of the present invention.

Claims

1. A SNP molecular marker combinatorial system for largemouth bass, characterized in that, The genome includes 6112 SNP molecular markers, and the information of these SNP molecular markers on the largemouth bass genome version GCA_224357851 is as follows: 。 2. A method for screening SNP molecular marker combinations in largemouth bass, characterized in that the steps include: include: (1) Whole genome resequencing was performed on all largemouth bass samples that underwent heat stress and pathogen infection experiments to obtain whole genome sequence information, and phenotypic data of heat tolerance, disease resistance, morphological traits and growth traits of all largemouth bass samples were statistically analyzed. (2) The whole genome sequence information was compared with the largemouth bass genome version GCA_224357851, SNP molecular markers were extracted, and whole genome resequencing data were obtained; (3) Perform genome-wide association analysis on the whole genome resequencing data and the phenotypic data of heat resistance, disease resistance, morphological traits and growth traits to distinguish the core SNP molecular markers and background SNP molecular markers related to the phenotypic data. Based on the fact that the background SNP molecular markers uniformly cover the whole genome and there are no duplicate core SNP molecular markers within 50kb upstream and downstream of the background SNP molecular markers, at least 6112 SNP molecular markers as described in claim 1 are obtained.

3. An SNP molecular probe set, characterized in that, The SNP molecular probe set contains SNP molecular probes that target at least the 6112 SNP molecular markers described in claim 1.

4. The application of the SNP molecular probe set according to claim 3 in the preparation of whole genome SNP chip products of largemouth bass.

5. A whole-genome SNP chip for largemouth bass, characterized in that, It contains an SNP molecular probe set as described in claim 3.

6. A reagent kit, characterized in that, It contains an SNP molecular probe set as described in claim 3 and / or a whole genome SNP chip of largemouth bass as described in claim 5.

7. The use of at least one of the largemouth bass whole genome SNP chip according to claim 5 and / or the kit according to claim 6 in the identification of largemouth bass germplasm resources, genetic diversity analysis, genome-wide association analysis, mining of important functional genes and selection breeding.

8. The use according to claim 7, characterized in that, The selective breeding is selected from at least one of the following: variety identification, parentage identification, population identification, pedigree reconstruction, inbreeding level assessment, optimized mating scheme, fixation of superior genes, trait assessment and prospect selection.

9. A method for obtaining the whole genome type of largemouth bass, characterized in that the steps include... include: (1) Extract genomic DNA from largemouth bass samples and construct sequencing libraries; (2) Use the whole genome SNP chip of largemouth bass as described in claim 5 and / or the kit as described in claim 6 to perform hybridization capture to obtain the captured fragment; (3) High-throughput sequencing was performed using the captured fragments, and the results were compared with the reference genome of the largemouth bass to obtain the genotyping data of the whole genome of the largemouth bass.

10. A method for the selection and evaluation of largemouth bass, characterized by the following steps: include: Based on the acquisition method described in claim 9, the genotyping data of the largemouth bass statistical samples are obtained. A largemouth bass breeding value prediction model is constructed using a genomic breeding value statistical model. The largemouth bass test sample is input based on the genotyping data obtained by the acquisition method described in claim 9 to obtain the breeding value of the largemouth bass test sample and to conduct a largemouth bass breeding evaluation.

Images