A megalobrama amblycephala whole genome liquid chip and a preparation method and application thereof

Abstract

This invention discloses a whole-genome liquid-phase microarray of blunt snout bream, its preparation method, and its applications. The microarray contains 49,173 highly polymorphic core SNP molecular markers, the loci of which were determined by sequence alignment with reference to the blunt snout bream genome ASM1881202v1. The whole-genome liquid-phase microarray of blunt snout bream prepared in this invention selects characteristic SNP loci from three major wild populations and three new varieties, ensuring that the loci are evenly distributed across the genome and comprehensively cover core variation information under different genetic backgrounds, giving the markers broad universality and effectiveness. Simultaneously, the microarray includes loci significantly associated with important economic traits such as growth, disease resistance, and hypoxia tolerance, closely integrating molecular markers with actual production needs. It can not only be used to detect the genotype of blunt snout bream DNA samples but also directly serve population cluster analysis and the breeding of disease-resistant new varieties, significantly enhancing the practical value of the microarray in blunt snout bream breeding practice.

Description

Technical Field

[0001] This invention relates to the field of gene chip technology, specifically to a whole genome liquid-phase chip of blunt snout bream, its preparation method, and its application. Background Technology

[0002] The bluntnose bream (Megalobrama amblycephala), belonging to the order Cypriniformes, family Cyprinidae, and genus Megalobrama, is also known as the Wuchang bream. However, with the expansion of aquaculture, problems such as germplasm degradation, uneven individual size, slowed growth rate, frequent diseases, and premature sexual maturity have become increasingly prominent. Although three new bluntnose bream varieties targeting growth and hypoxia tolerance have been developed, high-quality germplasm resources remain scarce, and research on breakthrough varieties with disease resistance and stress tolerance is extremely weak. There is a need to innovate breeding technology systems and utilize efficient modern molecular breeding techniques to cultivate new varieties that are fast-growing, disease-resistant, stress-resistant, and have excellent meat quality, promoting the sustainable and healthy development of the bluntnose bream industry and further improving the economic and social benefits of bluntnose bream aquaculture.

[0003] Genomic selection (GS) is an effective molecular breeding technique. The accuracy of GS relies on SNP loci covering the entire genome, and high-throughput and high-quality genotyping technology has always been a bottleneck for GS. Currently, the mainstream high-throughput genotyping methods include next-generation sequencing (NGS) and gene chips. NGS is costly for genotyping large batches of samples. In comparison, gene chips are a more economical and efficient genotyping method. Gene chips can be divided into solid-phase and liquid-phase types. Liquid-phase chip technology, developed in the mid-1990s, is hailed as the chip technology of the post-genomic era. Liquid-phase chips, especially those based on targeted sequencing genotyping, not only possess the advantages of high efficiency, accuracy, and reproducibility of solid-phase chips but also offer flexible marker design and low genotyping costs, making them one of the most popular high-throughput genotyping platforms. Currently, liquid-phase breeding chips have been successfully developed and widely applied in more than 20 crops, including rice, cotton, and corn, as well as livestock and poultry such as pigs, chickens, cattle, and sheep.

[0004] In the aquaculture sector, high-throughput genotyping chips for assisted breeding have been developed for species such as turbot, large yellow croaker, golden pomfret, grouper, and oysters. Currently, there are no reports on breeding chips for blunt snout bream, either domestically or internationally. Summary of the Invention

[0005] The main objective of this invention is to propose a whole-genome liquid microarray for blunt snout bream, its preparation method, and its application. The aim is to provide a liquid microarray for blunt snout bream that covers the entire genome, has high throughput, high flexibility, and low cost per marker, providing a technical means for assessing genetic diversity, identifying germplasm resources and kinship relationships, conducting genome-wide association studies, and selecting and breeding for important economic traits, thus meeting the needs of large-scale whole-genome breeding of blunt snout bream.

[0006] To achieve the above objectives, this invention proposes a whole-genome liquid-phase microarray for blunt snout bream, which contains 49,173 highly polymorphic core SNP molecular markers. The site information of these 49,173 highly polymorphic core SNP molecular markers is shown in Table 1 below:

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[0112] The 49,173 highly polymorphic core SNP molecular marker sites were determined by sequence alignment with reference to the blunt snout bream genome ASM1881202v1.

[0113] Preferably, the 49,173 highly polymorphic core SNP molecular markers include 47,056 background SNP sites and 2,117 functional SNP sites.

[0114] Preferably, the 49,173 highly polymorphic core SNP molecular markers are associated with important economic traits of blunt snout bream, including one or more of growth, disease resistance, sex, gonadal development, hypoxia tolerance, muscle development, diet, and intermuscular spines.

[0115] Preferably, the liquid-phase chip further includes probes designed based on gene sequences covering SNP molecular sites.

[0116] This invention also proposes a method for preparing the whole genome liquid-phase chip of blunt snout bream as described above, comprising the following steps:

[0117] (1) Screening for high-quality SNP sites from resequencing data of blunt snout bream;

[0118] (2) Based on previous multi-omics data of blunt snout bream, 1800 genes, 10 RAD-seq markers and 7 SNP sites were screened that are associated with traits such as growth, disease resistance, sex, gonadal development, hypoxia tolerance, muscle development, diet and intermuscular spines.

[0119] (3) By comparing the trait-associated genes with the RAD-seq data of the blunt snout bream reference genome (ASM1881202v1), SNP sites in the region were identified.

[0120] (4) Filter out SNP sites in the region that have MAF < 0.01, and calculate the linkage disequilibrium R between sites. 2 Value, retain R 2 Strong linkage sites with a value >0.8 were used to screen for trait-associated SNPs, taking into account site location and mutation type.

[0121] (5) Divide the genome into high-quality SNPs obtained by resequencing using a 20kb sliding window, calculate haplotypes and screen high-value sites based on information weights;

[0122] (6) The uncovered window was split into 5kb for supplementary design, and finally 49,173 highly polymorphic core SNP sites from two sources (whole genome + functional region) were integrated;

[0123] (7) Based on the probe design criteria and by bowtie2 end-to-end alignment, sites that cannot be synthesized with high-quality probes are removed, and finally a probe pool containing two types of markers is formed, namely the whole genome liquid chip of blunt snout bream.

[0124] In step (2), the multi-omics data includes comparative transcriptomics, small RNA omics, and genome-wide association analysis.

[0125] This invention also proposes an application of the whole genome liquid phase chip of blunt snout bream as described above in the breeding of blunt snout bream.

[0126] This invention also proposes the application of the above-described whole genome liquid phase chip of blunt snout bream in detecting the genotype of blunt snout bream DNA samples.

[0127] This invention also proposes an application of the whole genome liquid phase chip of blunt snout bream as described above in population clustering analysis.

[0128] This invention also proposes an application of the whole genome liquid-phase chip of blunt snout bream as described above in genome-wide association analysis of disease resistance traits.

[0129] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0130] (1) The whole genome liquid-phase chip of blunt snout bream prepared in this invention selects characteristic SNP loci from three major wild populations and three new varieties to ensure that the loci are evenly distributed on the genome and can comprehensively cover the core variation information under different genetic backgrounds, so that the markers have broad universality and effectiveness. At the same time, the chip includes loci that are significantly associated with important economic traits such as growth, disease resistance, and hypoxia tolerance, which closely integrates molecular markers with actual production needs. It can not only be used to detect the genotype of blunt snout bream DNA samples, but also directly serve population cluster analysis and the breeding of disease-resistant new varieties, significantly improving the practical value of the chip in blunt snout bream breeding practice.

[0131] (2) This liquid-phase chip, relying on targeted capture sequencing technology, achieves efficient genotyping of target SNP loci, significantly simplifying the bioinformatics analysis process. Compared with traditional technologies, its detection efficiency is significantly improved, enabling rapid and low-cost genotyping of large-scale samples, effectively solving the problems of high cost and limited throughput of whole-genome resequencing. In addition, while genotyping the target locus, the chip can also capture and analyze SNPs in the surrounding area of ​​the target locus, obtaining more genetic information than marker loci, further enhancing the richness of the data and the accuracy of the analysis, providing more comprehensive molecular data support for the genetic breeding research of blunt snout bream.

[0132] (3) Compared with traditional solid-phase chips, the liquid-phase chip of this invention has greater flexibility, allowing for the addition or adjustment of marker sites at any time according to breeding needs, meeting the dynamic changes of different research directions and breeding objectives. Compared with whole-genome resequencing, it has a significant price advantage, enabling large-scale genotyping of blunt snout bream populations at a lower cost, with high genotyping accuracy, greatly reducing the technical threshold and economic cost of molecular breeding. The successful development of this chip provides a powerful technical tool for the identification of blunt snout bream germplasm resources, phylogenetic analysis, and genome-wide association studies, effectively promoting the transformation of blunt snout bream breeding from traditional phenotypic selection to precision molecular design breeding, promoting the sustainable and healthy development of the industry, and significantly improving the economic and social benefits of aquaculture. Attached Figure Description

[0133] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0134] Figure 1 This is a schematic diagram showing the distribution of the core sites of the blunt snout bream liquid phase chip in the genome of the present invention.

[0135] Figure 2This is a graph showing the SNP site detection rate of the liquid phase chip for blunt snout bream according to the present invention.

[0136] Figure 3 This is a bar chart showing the statistical consistency rate between the liquid phase chip and resequencing genotyping of the blunt snout bream of this invention.

[0137] Figure 4 Phylogenetic tree (A) constructed based on the SNP genotyping results of the blunt snout bream liquid phase chip of this invention and phylogenetic tree (B) constructed based on the SNPs obtained from resequencing analysis.

[0138] Figure 5 The figure shows the correlation analysis results of disease resistance traits in blunt snout bream based on the liquid phase chip of the present invention.

[0139] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0140] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0141] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0142] Example 1: Preparation of whole genome liquid-phase chip of blunt snout bream

[0143] (1) Whole genome resequencing of blunt snout bream population and acquisition of high-quality SNP sites

[0144] To develop a liquid-phase breeding chip for blunt-snout bream, this invention first constructs a basic data pool through multi-population whole-genome resequencing of blunt-snout bream. The study selected 270 individuals from nine populations, including wild populations from Liangzi Lake, Poyang Lake, and Yuni Lake, and nationally approved varieties such as “Pujiang No. 1,” “Huahai No. 1,” and “Pujiang No. 2,” for whole-genome resequencing analysis. Through a rigorous screening process, 16,836,560 highly reliable high-quality SNP loci were obtained. The locus distribution is shown below. Figure 1As shown, the screening of high-quality SNP sites is based on the following principles: (1) The SNP site is located in a non-repetitive region of the genome and has only two base forms, with each base form accounting for a certain proportion. (2) SNP sites with a deletion rate higher than 5% and a minor allele frequency (MAF) of less than 0.01 are filtered out.

[0145] (2) Screening of functional loci and core markers related to important traits in blunt snout bream

[0146] By combining genome-wide data with association analysis of economic traits, functional markers were enriched in a targeted manner. Based on previous findings in blunt snout bream genomics, this study collected 1800 genes, 10 RAD-seq sites, and 7 SNP loci associated with growth, disease resistance, sex, gonadal development, hypoxia tolerance, muscle development, diet, and intermuscular spine traits. Core markers were then selected through functional region SNP locus design and genome-wide marker simplification.

[0147] Design of SNP loci in functional regions: Based on 1800 genes, 10 RAD-seq results, and 7 SNP loci accumulated from previous omics studies related to traits, trait-related SNP loci were first identified by aligning the trait-related RAD-seq and gene sequences to the *Brucea bream* reference genome (ASM1881202v1). Loci with a MAF < 0.01 were removed, and then linkage disequilibrium R0.01 was calculated for any two loci within the region. 2 Value, set R 2 A value >0.8 indicates strong linkage. Simultaneously, filtering was performed based on minimum allele frequency (MAF), the relative position of the SNP to the midpoint, and the mutation type to obtain trait-related SNP sites for candidate probe design. For gene regions and RAD-seq regions where probe design was unsuccessful, SNP sites strongly linked to the SNPs within the region were identified through strong linkage relationships for supplementary design, ultimately yielding trait-related SNP sites.

[0148] Genome marker simplification: First, based on the 16,836,560 high-quality SNPs obtained from resequencing, the *Brucea buergeriana* genome was divided into 55K window regions of 20kb each. Haplotypes for each population were calculated using a 20kb sliding window. Based on the weights of haplotypes and loci (refer to Table 2 for weight setting standards), the loci with the highest weight information content were selected as candidate sites for subsequent probe design. At the same time, supplementary designs were made for windows that could not be designed for probes. These windows were further divided into 5kb regions, and haplotypes within each window were calculated. The SNP with the highest score within each haplotype was selected for probe design.

[0149] Table 2 SNP Weight Information Table

[0150]

[0151] Through the above process, 49,173 highly polymorphic core SNP sites were finally obtained from two sources: whole genome resequencing and functional regions. Specifically, these include 47,056 background SNP sites and 2,117 functional SNP sites, which serve as the core marker library for probe design. The chip site information is shown in Table 1.

[0152] (3) Design and fabrication of SNP liquid-phase chip for blunt snout bream

[0153] Based on the core marker library obtained through screening, a liquid-phase breeding chip for the whole genome of blunt-snout bream was systematically designed and prepared. According to probe design standards, the probe length was 120 nt, the GC content was controlled at 20-80%, and the annealing temperature was 40-200℃. Then, bowtie2 end-to-end alignment was used to ensure the uniqueness of the probe sequences and the absence of short repeats. SNP sites that could not be used to design high-quality liquid-phase probes were removed. The resulting probe pool is the whole genome liquid-phase breeding chip for blunt-snout bream. After the above process, a probe pool containing two types of core SNP sites (those from whole-genome resequencing and those related to functional traits) is formed, which is the whole genome liquid-phase breeding chip for blunt-snout bream.

[0154] Example 2: Application of Blunt-snout Bream Liquid Chromatography Chip in Detecting the Genotype of Blunt-snout Bream DNA Samples

[0155] Genotyping of 36 bluntnose bream was performed using a developed liquid-phase breeding chip for bluntnose bream. The specific procedure is as follows:

[0156] (1) Genomic DNA extraction and detection from blunt snout bream: Genomic DNA was extracted from the fin tissues of 36 blunt snout bream using a high-throughput DNA kit. The purity and integrity of the DNA were examined using 1% agarose gel electrophoresis, and the DNA concentration was accurately quantified using Qubit.

[0157] (2) The DNA sample that has passed the test is sonicated and broken. After the ends are repaired, the A tail is connected and connected to the sequencing adapter. Then, the library is purified and fragments are selected using carboxyl-modified magnetic beads. PCR amplification is performed using sequencing primers with barcodes and a high-fidelity PCR reaction system. The amplified product after purification by carboxyl magnetic beads can be used for probe hybridization experiments.

[0158] (3) After washing the hybridization products with washing buffer, a second round of PCR was performed to complete the construction of the hybridization capture library. After the library construction was completed, preliminary quantification was performed using Qubit 2.0, and the effective concentration of the library was accurately quantified using real-time PCR to ensure library quality. After 36 sample libraries passed the test, they proceeded to the sequencing stage.

[0159] (4) Information analysis process: Data quality control (removal of adapters and low-quality data), alignment with the reference genome, variant detection and annotation, etc., are used to obtain genotyping results. Genotyping results of 36 samples show that the detection rate of target SNP sites is between 98.13% and 99.91%. Figure 2 The detection rate was high. Simultaneously, these 36 samples underwent resequencing and genotyping. Comparison between the genotyping results from the microarray and the resequencing results showed a genotyping concordance rate between 96.95% and 98.86%. Figure 3 This indicates that the chip has high accuracy in high-throughput genotyping.

[0160] Example 3: Application of Blunthead Bream Liquid Phase Chip in Population Cluster Analysis

[0161] Genotyping of 52 blunt-snout bream from wild populations including Liangzi Lake (LZ), Poyang Lake (PY), and Yuni Lake (YN), as well as new varieties such as "Pujiang No. 1" (PJ1), "Huahai No. 1" (HH1), and "Pujiang No. 2" (PJ2), was performed using the chip of this invention. Simultaneously, whole-genome resequencing was used for genotyping. Phylogenetic trees were then constructed based on the genotyping results of both methods. The phylogenetic trees constructed from both genotyping results showed that individuals from the Yuni Lake, Pujiang No. 1, Huahai No. 1, and Pujiang No. 2 populations clustered into separate branches, while the Liangzi Lake and Poyang Lake populations showed mixed populations. Figure 4 The results show that the phylogenetic tree of the blunt snout bream population constructed based on the chip typing results is very close to the phylogenetic tree constructed based on the whole genome resequencing typing results, and the chip can be applied to the cluster analysis of the blunt snout bream population.

[0162] Example 4: Application of Blunt-headed Bream Liquid Phase Microarray in Genome-wide Association Analysis of Disease Resistance Traits

[0163] A population of blunt snout bream was challenged with Aeromonas hydrophila via intraperitoneal injection. Fin samples were collected from 99 dead (susceptible) and 106 surviving (resistant) individuals. A binary survival phenotype was used to represent two categories: 1 for resistant individuals and 2 for susceptible individuals. Genotyping of these 205 individuals was performed using a microarray (see Example 2). Quality control was performed on the genotyping results, and high-quality genotyping information was selected. Individuals with a minimum allele frequency below 5%, a genotype loss rate greater than 5%, and a sample loss rate greater than 2% were removed because these loci and individuals had insufficient data reliability and completeness, making them unsuitable for further data analysis. After screening, 67,199 SNP markers and 205 individuals were obtained. Genome-wide association analysis of the disease resistance trait was conducted based on a mixed linear model (MLM), and the Bonferroni test was used to screen for significant SNP loci, ultimately identifying 10 SNP loci significantly associated with the disease resistance trait. Figure 5 The study identified eight genes, including phldb2a, LOC125262741, smyd4, ubl3a, rsf1b, and acsf2. Most of these candidate genes have been reported to play important roles in the immune process, providing important basic data for disease-resistant breeding research in blunt snout bream.

[0164] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A liquid-phase microarray containing the whole genome of a blunt-snout bream, characterized in that, The chip contains 49,173 highly polymorphic core SNP molecular markers, and the site information of these 49,173 highly polymorphic core SNP molecular markers is shown in the table below: The 49,173 highly polymorphic core SNP molecular marker sites were determined by sequence alignment with reference to the blunt snout bream genome ASM1881202v1.

2. The whole genome liquid-phase chip of blunt snout bream according to claim 1, characterized in that, The 49,173 highly polymorphic core SNP molecular markers include 47,056 background SNP sites and 2,117 functional SNP sites.

3. The whole genome liquid chip of Megalobrama amblycephala according to claim 1, characterized in that, The 49,173 highly polymorphic core SNP molecular markers are associated with important economic traits of blunt snout bream, including one or more of the following: growth, disease resistance, sex, gonadal development, hypoxia tolerance, muscle development, diet, and intermuscular spines.

4. The whole-genome liquid chip of Megalobrama amblycephala according to claim 1, characterized in that, The liquid-phase chip also includes probes designed based on gene sequences covering SNP molecular sites.

5. A method for preparing a whole genome liquid chip of Megalobrama amblycephala according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Screening for high-quality SNP sites from the resequencing data of blunt snout bream; (2) Based on the previous multi-omics data of blunt snout bream, 1800 genes, 10 RAD-seq markers and 7 SNP sites were screened that are associated with traits such as growth, disease resistance, sex, gonadal development, hypoxia tolerance, muscle development, diet and intermuscular spines. (3) By comparing the trait-associated genes with the RAD-seq data to the reference genome of blunt snout bream ASM1881202v1, the SNP sites in the region were identified; (4) Filter out the SNP sites with MAF < 0.01 in the region, calculate the linkage disequilibrium R 2 value between sites, keep the strong linkage sites with R 2 > 0.8, combine site position and mutation type, and screen trait-associated SNPs; (5) Divide the genome into 20 kb sliding windows for the high-quality SNPs obtained by resequencing, calculate haplotypes and screen high-value sites based on information weights; (6) The uncovered window was split into 5 kb for supplementary design, and finally 49,173 highly polymorphic core SNP sites from two sources, including the whole genome and functional regions, were integrated; (7) Based on the probe design criteria and by bowtie2 end-to-end alignment, sites that cannot be synthesized with high-quality probes are removed, and finally a probe pool containing two types of markers is formed, namely the whole genome liquid chip of blunt snout bream.

6. The application of the whole genome liquid phase chip of blunt snout bream as described in any one of claims 1-4 in the breeding of blunt snout bream.

7. The application of the whole genome liquid phase chip of blunt snout bream as described in any one of claims 1-4 in detecting the genotype of blunt snout bream DNA samples.

8. An application of the whole genome liquid phase chip of blunt snout bream as described in any one of claims 1-4 in population cluster analysis.

9. The application of the whole genome liquid phase chip of blunt snout bream as described in any one of claims 1-4 in genome-wide association analysis of disease resistance traits.

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