A large mouth bass 100k liquid chip and its application

Abstract

This invention belongs to the field of gene chip technology, specifically relating to a 100k liquid phase chip for largemouth bass and its applications. The SNP liquid phase chip of this invention contains 102,075 SNP molecular markers and 102,065 capture probes. The probe combinations are used to identify the genotype of each SNP locus in the SNP locus combination. The high-density SNP liquid phase chip for largemouth bass provided by this invention can rapidly and accurately detect the genotype of the sample, with an average SNP detection rate of 99.73% and an average genotype consistency rate of 99.42%. It exhibits good genotyping stability and high accuracy. The chip prepared by this invention can be widely used for gene mining of important economic traits in largemouth bass, QTL mapping, and genome-wide selection breeding, effectively improving the efficiency and accuracy of largemouth bass breeding.

Description

Technical Field

[0001] This invention belongs to the field of gene chip technology, specifically relating to a 100k liquid phase chip for largemouth bass and its applications. Background Technology

[0002] Liquid-phase chips, also known as suspension arrays (liquid chips), are a novel biochip technology platform based on xMAP (flexible multi-analyte profiling) technology. They enable the binding reactions of antigens and antibodies, enzyme substrates, ligands and receptors, and nucleic acid hybridization reactions on microspheres with different fluorescent codes. Qualitative and quantitative results are achieved by detecting the microsphere coding and reporter fluorescence using red and green lasers, respectively. Up to 100 different biological reactions can be completed within a single reaction well, making it a next-generation high-throughput molecular detection technology platform following gene chips and protein chips.

[0003] Hundreds of xMAP-based detection platforms worldwide are used in fields such as immunology, protein and nucleic acid detection, and gene research. This technology has become a new tool for proteomics and genomics research.

[0004] Liquid phase microarrays have been developed for crops such as rice and corn, livestock and poultry such as pigs and chickens, and aquatic animals. More than 50 sets of liquid phase microarrays have been developed and have achieved relatively mature applications in species evolution analysis, germplasm resource evaluation and DNA fingerprinting, molecular genetic mapping, gene / QTL localization and gene cloning, marker-assisted selection, and whole-genome selection. For example, the applicant's Chinese invention patent application (publication number: CN117089624A, publication date: 2023-11-21) discloses a whole-genome SNP liquid phase microarray for small yellow croaker and its application, which relates to the field of biotechnology. The small yellow croaker whole-genome SNP liquid phase microarray includes a probe array used to identify the genotype of each SNP locus in the SNP locus array. The invention provides a whole-genome SNP liquid phase chip for small yellow croaker, in which SNP sites are evenly distributed across the entire genome chromosome with high coverage. It can be applied to genomic selection breeding of important economic traits such as growth and disease resistance in small yellow croaker. Using the whole-genome SNP liquid phase chip for small yellow croaker of this invention, genotyping of small yellow croaker samples can be performed quickly and applied to genomic selection breeding, which helps to shorten the breeding cycle of small yellow croaker and accelerate the process of breeding new varieties of small yellow croaker.

[0005] Largemouth bass (Micropterus salmoides), also known as California bass, has rapidly become an important freshwater aquaculture species due to its delicious meat, lack of intramuscular bones, rapid growth, and short breeding cycle. In recent years, the scale of largemouth bass farming has been continuously expanding, and the degree of intensification has been increasing. However, problems such as limited growth potential, frequent disease outbreaks, and rising farming costs have also arisen. Utilizing efficient modern molecular breeding techniques to cultivate new fast-growing, high-yielding, and disease-resistant largemouth bass varieties can effectively address these problems and their harm to the largemouth bass aquaculture industry. Therefore, there is an urgent need to develop a high-density SNP chip for germplasm improvement of important economic traits in largemouth bass, enabling early selection, accelerating genetic progress, shortening generation intervals, and effectively improving the efficiency and accuracy of largemouth bass breeding. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a 100k SNP liquid phase chip for largemouth bass and its applications. The chip loci are evenly distributed on the genome chromosome, with high coverage, good polymorphism, and strong versatility, and can be effectively used for largemouth bass genetic background identification, gene mining, and breeding.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A 100k liquid phase chip for largemouth bass contains probes corresponding to SNP site combinations; the SNP site combinations consist of 102,075 SNP molecular markers, and the site information is shown in Table 1.

[0009] Table 1. Locatory information for largemouth bass

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[0163] Furthermore, the present invention also discloses a probe combination for identifying the genotype of each SNP site in the SNP site combination.

[0164] Furthermore, the present invention also discloses the application of the liquid phase chip or the probe combination in screening for major economic traits of largemouth bass, wherein the economic traits include one or more of growth, disease resistance, sex, and stress resistance.

[0165] Furthermore, the present invention also discloses the application of the liquid phase chip or the probe combination in the genome selection breeding of largemouth bass.

[0166] Furthermore, the present invention also discloses a method for whole-genome genotyping detection of largemouth bass, comprising: extracting genomic DNA from a largemouth bass sample to be tested; detecting the genomic DNA using the liquid phase chip to obtain raw data; and performing data analysis on the raw data to obtain genotyping results.

[0167] Furthermore, the present invention also discloses the application of the method in screening for major economic traits of largemouth bass, wherein the economic traits include one or more of growth, disease resistance, sex, and stress resistance.

[0168] Furthermore, the present invention also discloses the application of the method in genomic selection breeding of largemouth bass.

[0169] Furthermore, the present invention also discloses the application of the liquid phase chip, the probe combination, or the method described herein in the precise identification and evaluation of largemouth bass germplasm resources and varieties, the construction of high-density genetic maps, QTL mapping, key gene mining, or whole-genome selection breeding.

[0170] The 100k liquid phase chip for largemouth bass provided by this invention can rapidly and accurately detect the genotype of largemouth bass individuals, with an average SNP detection rate of 99.73% and an average genotype concordance rate of 99.42%. It exhibits good genotyping stability, high accuracy, and excellent overall performance. The 100k liquid phase chip provided by this invention contains significant loci associated with traits such as growth, sex, temperature, and immunity. The largemouth bass whole-genome SNP liquid phase chip prepared using this invention can rapidly genotype largemouth bass samples, requiring only a small number of samples and having no sample quantity limitation. This chip can be widely used for precise identification and evaluation of largemouth bass germplasm resources and varieties, construction of high-density genetic maps, QTL mapping, key gene mining, and whole-genome selection breeding, providing important technical support for the breeding of superior largemouth bass breeds and effectively improving the efficiency and accuracy of largemouth bass breeding. Attached Figure Description

[0171] Figure 1 Distribution of 100k liquid-phase microarray marker sites on chromosomes of largemouth bass.

[0172] Figure 2 Distribution map of MAF at the 100k site of largemouth bass.

[0173] Figure 3 Detection rate of loci in 100k liquid phase chip for largemouth bass.

[0174] Figure 4 Genotypic consistency rate of duplicate samples from 100k liquid phase microarray of largemouth bass.

[0175] Figure 5 Manhattan plot of genome-wide association analysis based on 100k microarray genotyping results of largemouth bass. Detailed Implementation

[0176] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present invention.

[0177] Example 1

[0178] A 100k SNP liquid phase chip for largemouth bass is mainly obtained through the following process:

[0179] This invention collected the whole-genome genetic information of 110 individuals from three populations of largemouth bass (Taiwanese perch, Sichuan perch, and Guangxi perch) in China using whole-genome resequencing technology. Through rigorous screening, 102,075 qualified SNP loci were obtained, and 102,065 capture probes were synthesized. Finally, a high-density 100k SNP liquid-phase chip for largemouth bass was developed. The specific implementation is as follows:

[0180] (1) Whole genome resequencing of largemouth bass and identification and screening of SNP sites

[0181] Growth phenotypes (body weight, body length, total length, body height, and body width, etc.) and caudal fin samples were collected from 110 samples of different largemouth bass broodstock populations (Taiwanese perch, Sichuan perch, and Guangxi perch) from three groups. After DNA extraction and passing agarose gel electrophoresis and Nanodrop assays, whole-genome resequencing was used for sequencing analysis. Raw reads were quality controlled by removing reads with adapters, reads containing more than 5% N, and low-quality reads to obtain clean reads. BWA software was used to map the clean reads to the largemouth bass reference genome (ASM2 243578v1). Based on the alignment results, GATK software was used for variant detection. The filtered largemouth bass resequencing data underwent alignment analysis with the reference genome and variant site screening, resulting in 5.03 million SNP loci.

[0182] (2) Development of 100k SNP liquid phase chip for largemouth bass

[0183] Based on the online publications of English and Chinese articles on largemouth bass and the genetically related loci (SNPs) for growth, sex, immunity, and heat tolerance previously discovered by the inventors in largemouth bass, 561 relevant SNP loci were initially obtained after removing duplicate loci.

[0184] Genome-wide association analysis (GWAS) was performed using phenotypic and genotypic data of various economic traits. Based on indicators such as chromosomal location, minimum allele frequency (MAF), deletion rate (NA), and heterozygosity of relevant SNP loci, 102,075 superior SNP loci (containing 561 functional gene loci) were rigorously screened (Table 1). The chromosomal distribution of the 102,075 SNP loci is shown below. Figure 1 The distribution of MAF values ​​can be found in the following figures. Figure 2 Based on the principles of probe and primer design, 102,065 capture probes that uniformly cover the entire genome, exhibit high polymorphism, strong specificity, and good versatility were finally synthesized, resulting in a high-density 100k SNP liquid-phase chip for largemouth bass.

[0185] Example 2

[0186] Quality evaluation of genotyping of largemouth bass 100k SNP liquid phase chip

[0187] (1) Demo test results

[0188] Twelve largemouth bass samples were randomly selected and genotyping was performed using the largemouth bass 100k liquid phase chip described in Example 1. After sequencing and data analysis, the average detection rate of loci in the largemouth bass samples was 99.49%, and the average genotypic consistency rate with the resequencing samples was 95.03%.

[0189] (2) Production test results

[0190] Ninety-one new largemouth bass samples were randomly selected for 100k liquid-phase chip production testing. Sequencing and data analysis showed an average locus detection rate of 99.77%. Figure 3 The average genotypic similarity rate of the technically replicated samples was 99.57%. Figure 4 The test has a high genotype detection rate and accuracy, good stability, and excellent overall performance.

[0191] Example 3

[0192] Genome-wide association analysis of important economic traits in largemouth bass based on 100k liquid microarray

[0193] Genome-wide association analysis (GWAS) of growth traits (total length, body length, body height, and body weight) of largemouth bass was performed using the 100k liquid phase chip from Example 1. SNP loci from 1619 largemouth bass samples were screened using VCFtools based on a deletion rate ≤0.1 and a maximum mean squared error (MAF) ≥0.05 (max-missing 0.9 – MAF 0.05), resulting in 96,193 SNP loci from 1619 samples for GWAS analysis. Genome-wide association analysis of the four growth traits was performed using rMVP, employing three computational models: generalized linear model (GLM), mixed linear model (MLM), and FarmCPU model. Principal components were added as covariates to the models for correction. Genome-wide association analysis was performed on each of the four phenotypic traits. Figure 5 The results showed that SNP loci significantly associated with all four growth traits were identified, with loci 1 and 12 being the major linkage groups associated with these traits. Therefore, the 100k liquid-phase chip for largemouth bass described in this invention can obtain relatively accurate genome-wide association analysis results.

[0194] Example 4

[0195] Application of 100k liquid phase chip in breeding of largemouth bass

[0196] Genotyping of 1619 individuals from a largemouth bass breeding population was performed using the 100k liquid phase chip described in Example 1. The method is as follows:

[0197] (1) The tail fins of 1,619 largemouth bass were collected for DNA extraction;

[0198] (2) Operate and analyze data according to the standard testing procedure of cGPS liquid phase chip of Huazhi Biotechnology Co., Ltd.;

[0199] (3) After detection and analysis, the average detection rate of SNP loci in the samples was 99.66%, the average heterozygosity rate was 34.67%, and the average genotypic consistency rate of the duplicate samples was 99.50%. The GBLUP method was used to predict the individual genome estimated breeding value (GEBV) of growth traits such as total length, body length, body height, and body weight of largemouth bass. The model prediction accuracy was 0.2949 to 0.5251, indicating that the 100k liquid phase chip for largemouth bass has high reliability in whole-genome selection.

[0200] The foregoing description of embodiments of the present invention, through which those skilled in the art are able to implement or use the present invention, will be readily apparent to those skilled in the art. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novelty disclosed herein.

Claims

1. A 100k liquid phase chip for largemouth bass, the chip containing probes corresponding to SNP site combinations; the SNP site combinations consist of 102,075 SNP molecular markers, the site information of which is shown in Table 1 of the specification, and the reference genome of the largemouth bass is ASM2 243578v1.

2. A probe combination for identifying the genotype of each SNP site in the SNP site combination of claim 1.

3. The application of the liquid phase chip of claim 1 or the probe combination of claim 2 in screening and economic traits of largemouth bass, wherein the economic traits are the growth traits of largemouth bass in terms of total length, body length, body height and weight.

4. The application of the liquid phase chip of claim 1 or the probe combination of claim 2 in genome-wide association analysis and breeding of growth traits of largemouth bass in total length, body length, body height and weight.

5. A method of whole-genome genotyping of a largemouth bass comprising: Genomic DNA extraction from largemouth bass samples to be tested; The genomic DNA was detected using the liquid-phase chip described in claim 1 to obtain raw data; Data analysis was performed on the raw data to obtain genotyping results.

6. The application of the method of claim 5 in screening for economic traits of largemouth bass, wherein the economic traits are growth traits of largemouth bass in terms of total length, body length, body height, and body weight.

7. The application of the method described in claim 5 in genome-wide association analysis and breeding of growth traits of largemouth bass in terms of total length, body length, body height and weight.

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