SNP chip of channa maculata and application thereof

Abstract

The embodiment discloses a SNP chip of snakehead and application thereof. The SNP chip comprises a probe combination fixed with SNP1-SNP50000. The chip has uniform distribution of sites, high accuracy of typing, strong repeatability, high flexibility, and universality, and is suitable for mainstream second-generation sequencing platforms such as Illumina and MGI. The chip can provide reliable genotyping technology for related researches such as snakehead population genetics analysis, kinship identification, whole genome association analysis and genome selection, improve the breeding efficiency of snakehead, and promote the development of aquatic species industry.

Description

Technical Field

[0001] This application relates to the field of snakehead technology, specifically to the SNP chip of snakehead and its application. Background Technology

[0002] The spotted snakehead (Channa maculata) belongs to the order Perciformes, family Channidae, and genus Channa. Hybrid snakeheads are offspring of the black snakehead (Channa argus) and the spotted snakehead, exhibiting advantages such as rapid growth, strong disease resistance, and ease of consuming formulated feeds. The spotted snakehead can also serve as the parent stock for new varieties such as "Hang Snakehead No. 1," "Black Spotted Hybrid Snakehead," and "Male Snakehead No. 1." However, inbreeding, germplasm degradation, and frequent diseases hinder the sustainable and healthy development of snakehead farming. Therefore, conducting superior breeding of spotted snakeheads based on modern molecular breeding techniques is crucial. However, current molecular breeding research on spotted snakeheads still relies on whole-genome resequencing and simplified genome sequencing, lacking stable and efficient breeding microarrays. Summary of the Invention

[0003] This application is based on The 50K SNP breeding chip for the whole genome of snakehead, designed using targeted capture sequencing technology, has advantages such as high throughput, high flexibility, short cycle, and low cost. It can be applied to genetic breeding research such as snakehead germplasm resource evaluation, genome-wide association analysis, and genome selection, and can meet the needs of breeding superior snakehead varieties.

[0004] Based on this, this application discloses at least the following technical solutions:

[0005] In the first aspect, the embodiments disclose SNP marker combinations for genotyping of snakehead fish, including:

[0006] Secondly, the embodiments disclose an SNP chip. The SNP chip includes a combination of probes fixed to marks SNP1 to SNP50000 respectively.

[0007] Thirdly, the embodiments disclose the use of the SNP tag combination described in the first aspect or the SNP chip described in the second aspect.

[0008] Fourthly, the embodiments disclose a method for detecting biological samples, the method including detecting information of SNP1 to SNP50000 markers in the biological sample.

[0009] Fifthly, an embodiment discloses a method for constructing a snakehead DNA fingerprint database. The method includes: extracting genomic DNA from all snakehead species used to construct the snakehead DNA fingerprint database; and processing the genomic DNA of each snakehead species using the SNP chip described in the second aspect.

[0010] The detection of SNP1 to SNP50000 markers yields genotypic data for all the aforementioned snakehead species on the SNP1 to SNP50000 markers, thus forming a snakehead DNA fingerprint database.

[0011] Sixthly, the embodiments disclose a kit for constructing a snakehead DNA fingerprint database. The kit includes the SNP chip described in the second aspect and an instruction manual describing the method described in the fifth aspect.

[0012] In a seventh aspect, an embodiment discloses a method for analyzing the population structure of snakehead germplasm resources, comprising: extracting genomic DNA from all snakehead varieties to be analyzed for population structure; using the SNP chip described in claim 2 to detect SNP1 to SNP50000 markers on the genomic DNA of each snakehead, thereby obtaining genotype data of all snakeheads on the SNP1 to SNP50000 markers; and performing cluster analysis based on the genotype data to analyze the genetic structure of the snakehead.

[0013] Eighthly, an embodiment discloses a kit for taxonomic classification of snakehead germplasm resources. The kit includes the SNP chip described in the second aspect and an instruction manual describing the method described in the seventh aspect.

[0014] The SNP chip provided in this application contains SNP sites unique to the snakehead fish, exhibiting a high detection rate, higher genome coverage, and lower site deletion rate. This SNP chip can serve as a low-cost, short-cycle, high-quality genotyping tool. Its application is flexible; target SNP sites can be adjusted by adding or removing probes, making it highly valuable. Furthermore, this SNP chip is widely adaptable, compatible with various detection platforms, and requires no specific expensive equipment.

[0015] The 50,000 SNP loci are evenly distributed across the chromosome, exhibiting good detection rate and SNP polymorphism. This SNP chip demonstrates high genotyping accuracy and strong reproducibility, providing reliable support for genetic analysis of complex traits in snakehead and evaluation of germplasm resources, thereby improving breeding efficiency and promoting the development of the aquatic seed industry. Attached Figure Description

[0016] Figure 1 The distribution of 50,000 SNP sites on the snakehead genome provided in the example was set to a window of 10Kb.

[0017] Figure 2 The minimum allele frequency distribution map of 50,000 SNP loci provided for the examples ( Figure 2 A) SNP type distribution map Figure 2B) and statistical graphs of variations, coding regions, non-coding regions, introns, and exons ( Figure 2 C).

[0018] Figure 3 Manhattan scatter plot of genome-wide association analysis of weight trait in snakehead provided for example.

[0019] Figure 4 PCA diagram of the genetic structure analysis of the 67 wild-type tails provided for the example ( Figure 4 A) Phylogenetic tree diagram Figure 4 B) and group structure analysis diagram ( Figure 4 C). Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art. The terms "SNP," "SNP marker," or "SNP site" as used herein refer to nucleotide sequences present in the genomic sequence of a chromosome. Variations in polynucleotide sequences caused by differences in nucleotide sequences (changes in a single nucleotide—A, T, C, or G) result in chromosomal genome diversity, thereby allowing different alleles (e.g., alleles from two different individuals) or different individuals to be distinguished from each other. These variations may occur within coding or non-coding regions of a gene (e.g., promoter regions or their vicinity, or introns) or between genes. The term "allele" as used herein refers to different forms of the same gene present at a given locus on homologous chromosomes.

[0021] The term "linkage disequilibrium" as used in this article refers to a non-random association at two or more loci, which may be on the same chromosome or different chromosomes. Linkage disequilibrium is also known as gamete-level disequilibrium or gamete disequilibrium. In other words, linkage disequilibrium is the frequency in a population of alleles or genetic markers that are higher or lower than the frequency predicted by the random frequency of alleles. Linkage refers to a finite combination of two or more loci on a chromosome, while linkage disequilibrium is not the same as linkage. The degree of linkage disequilibrium depends on the difference between the observed and expected locus frequencies. For populations where the frequencies of recombination loci or genotypes equal the expected frequencies, we call it linkage equilibrium. The degree of linkage disequilibrium depends on many factors, including genetic linkage, selection, the probability of recombination, genetic drift, selective mating, and population structure.

[0022] As used in this article, "SNP chip" refers to a biochip that analyzes the presence of SNPs in a sample's DNA by arranging and attaching hundreds to hundreds of thousands of biomolecules as probes. These biomolecules, such as DNA, DNA fragments, cDNA, oligonucleotides, RNA, or RNA fragments with known sequences, are immobilized at specific intervals on a small solid substrate made of glass, silicon, nylon, or other organic materials. Depending on the degree of complementarity, hybridization occurs between the nucleic acids in the sample and the probes immobilized on the surface. By detecting and judging the hybridization, information about the substances contained in the sample can be obtained simultaneously.

[0023] SNP tagging combinations and SNP chips

[0024] The main types of SNP chips currently available include: in-situ synthesis on a substrate, which uses modified oligonucleotide monomers to synthesize spatially combined probe sequences stepwise in situ to form an SNP chip, thereby directly synthesizing an oligonucleotide probe array on a rigid surface; off-chip synthesis, which involves spotting pre-synthesized probe sequences onto specific sites to form an SNP chip, thereby creating a DNA probe array immobilized on a glass substrate; and microbead synthesis, which involves directly synthesizing DNA probes on encoded microbeads, or immobilizing pre-prepared probe sequences onto encoded microbeads, and then arbitrarily assembling them to form a microbead chip.

[0025] On one hand, the embodiments provide SNP marker combinations for genotyping of the spotted snakehead. The SNP marker combinations consist of SNP1 to SNP50,000 SNP markers. The SNP1 to SNP50,000 markers are represented sequentially in Table 1, for example, arranged sequentially from chromosome 1 to chromosome 21. After the SNP loci on each chromosome are arranged, they are then arranged sequentially on the chromosomes. The spotted snakehead reference genome is available in GenBank: GCA_020496755.1; assembly number: ASM2049675v1.

[0026] Table 1

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[0128] On one hand, an embodiment discloses an SNP chip. The SNP chip includes probe combinations immobilized with SNP1 to SNP50000 labels respectively. The probe combination consists of an oligonucleotide sequence formed by extending 120nt upstream and downstream of each SNP site in the SNP1 to SNP50000 labels. Preferably, the SNP site is located at the 60th or 61st nt position of the 120nt probe.

[0129] In some embodiments, the chip is fabricated using on-wafer in-situ synthesis, off-wafer synthesis, or microbead fabrication. In some embodiments, the chip is fabricated using in-situ photolithography, parallel photoresist layer synthesis, microfluidic channel on-wafer synthesis, light-guided in-situ synthesis, soft photolithography in-situ synthesis, inkjet printing, molecular imprint on-wafer synthesis, maskless chip fabrication, BeadArray, or suspended chip fabrication. In some embodiments, the chip is fabricated using Illumina Infinium technology or Affymetrix Axiom technology.

[0130] like Figure 1 As shown, the distribution of 50,000 SNP loci on the 21 chromosomes in the chip was statistically analyzed. It was found that all loci were evenly distributed across the 21 chromosomes of the snakehead fish, with an average inter-chromosome spacing of 12.38 kb. The SNP locus distribution map is shown below. Figure 1 .like Figure 2 The minimum allele frequency of SNP sites in the 50K SNP breeding chip of the whole genome of the spotted snakehead was statistically analyzed. The proportion of sites with a minimum allele frequency >0.1 was 97.95%, indicating a high proportion of polymorphic SNPs.

[0131] The genotyping performance of the chip was evaluated using 150 snakehead fish collected from Foshan, Guangdong, as a validation population. Whole-genome resequencing of the 150 snakehead fish and genotyping using the chip showed a 96.00% consistency between the two methods. Genotyping using the chip achieved a 97.94% SNP capture rate, indicating good genotyping results.

[0132] On the one hand, the embodiments disclose the uses of the above-mentioned SNP marker combinations or SNP chips. In some specific embodiments, the uses are selected from at least one of the following: (a) identification of snakehead germplasm resources; (b) identification of snakehead paternity; (c) SNP typing of snakehead; (d) identification or characterization of snakehead varieties; (e) molecular breeding of snakehead; (f) construction of snakehead DNA fingerprint database; (g) detection of snakehead variety purity; (h) genetic analysis of snakehead germplasm resources; (i) snakehead genome selection; (j) snakehead gene localization and cloning; (k) identification of snakehead functional genes; (l) construction of snakehead genetic maps; (m) genetic evolution and analysis; and (n) genome-wide association analysis.

[0133] On one hand, embodiments disclose a method for processing a biological sample. The method includes detecting information about SNP1 to SNP50000 markers in the biological sample. In some embodiments, the detection is performed using an SNP chip comprising probe combinations immobilized for SNP1 to SNP50000 markers, as shown in Table 1 (probes 1 to 50000).

[0134] One embodiment discloses a method for constructing a snakehead DNA fingerprint database. The method includes: extracting genomic DNA from all snakeheads used to construct the snakehead DNA fingerprint database; and using the SNP chip to detect SNP1 to SNP50000 markers on the genomic DNA of each snakehead, thereby obtaining genotype data of all snakeheads on the SNP1 to SNP50000 markers, thus constituting the snakehead DNA fingerprint database.

[0135] One embodiment discloses a kit for constructing a snakehead DNA fingerprint database. The kit includes the SNP chip and an instruction manual describing a method for constructing the snakehead DNA fingerprint database.

[0136] On one hand, the embodiments disclose a method for analyzing the population genetic structure of snakehead germplasm resources. The method includes extracting genomic DNA from the snakehead samples to be analyzed; using the SNP chip to detect SNP1 to SNP50000 markers on the genomic DNA of each snakehead, thereby obtaining genotype data for all snakeheads on the SNP1 to SNP50000 markers; and performing cluster analysis based on the genotype data to analyze the population genetic structure of all snakeheads.

[0137] On one hand, the embodiments disclose a kit for analyzing the population genetic structure of snakehead fish. The kit includes the SNP chip and instructions describing a method for analyzing the population genetic structure of snakehead fish.

[0138] Application of 50K SNP microarray in genome-wide association analysis of snakehead fish

[0139] Forty-four snakehead fish samples were collected, their weight data were measured, and genotyping was performed using the aforementioned microarray. The genotyping results were quality controlled using PLINK software, removing individuals with a minimum allele frequency <0.05, genotype deletion rate >0.10, or sample deletion rate >10%, resulting in 47,471 high-quality SNP loci. Genome-wide association analysis of the weight trait was performed using a mixed linear model in EMMAX software, and false positive SNP loci were removed using the Bonferroni test. Finally, 13 SNP loci associated with the snakehead fish's weight trait were located, distributed on chromosomes 2, 6, and 20. Figure 3 Through gene annotation, genes involved in cell proliferation, differentiation, migration, and metabolic regulation, among other growth-related genes, were successfully annotated. Therefore, this microarray can be used for genotyping of snakehead fish and can yield relatively accurate genome-wide association analysis results.

[0140] Population structure analysis and kinship identification

[0141] Genotyping of 67 wild snakehead fish from seven regions (Nanning, Shaoguan, Meizhou, Huazhou, Guilin, Shaowu, and Hengyang) was performed using a 50K SNP breeding chip across the entire snakehead genome. The genotyping results were quality-controlled using PLINK software, and population genetic structure analysis and phylogenetic identification were conducted. The results showed that the analytical results were consistent with the actual population genotyping results. Figure 4 This chip can be used to evaluate the germplasm resources of snakehead fish.

[0142] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. An SNP chip, comprising probe arrays immobilized with markers for SNP1 to SNP50000 respectively; the positions of the SNP1 to SNP50000 markers on the reference genome of *Channa argus* are shown in the table below, the GenBank accession number of the reference genome is GCA_020496755.1, and the assembly number is ASM2049675v1; the probe array consists of oligonucleotide sequences formed by extending 120 nt upstream and downstream of each SNP site in the SNP1 to SNP50000 markers, wherein the SNP site is located at the 60th or 61st nt position of the 120 nt probe. ； . ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； 。 2. The use of the SNP chip as described in claim 1, wherein the use is selected from at least one of the following (a) to (n): (a) Identification of snakehead germplasm resources; (b) Identifying the phylogenetic relationships of snakehead fish; (c) SNP typing of snakehead fish; (d) Identify or characterize species of snakehead fish; (e) Molecular breeding of snakehead fish; (f) Constructing a DNA fingerprint database for snakehead fish; (g) Detecting the purity of the spotted snakehead species; (h) Genetic analysis of snakehead germplasm resources; (i) Genomic selection in snakehead; (j) Gem mapping and cloning of the spotted snakehead; (k) Identification of functional genes in snakehead fish; (l) Construction of genetic map of snakehead fish; (m) Genetic evolution and analysis; (n) Genome-wide association analysis.

3. A method for constructing a snakehead DNA fingerprint database, comprising: Genomic DNA was extracted from all snakehead species used to construct the snakehead DNA fingerprint database. Using the SNP chip described in claim 1, the genomic DNA of each of the snakehead species is detected by SNP1 to SNP50000 markers, thereby obtaining the genotype data of all the snakehead species on the SNP1 to SNP50000 markers, thus forming a snakehead DNA fingerprint database.

4. A method for analyzing the population structure of snakehead germplasm resources, comprising: Genomic DNA was extracted from all snakehead species to be analyzed for population structure. Using the SNP chip described in claim 1, the genomic DNA of each snakehead is analyzed for SNP1 to SNP50000 markers, thereby obtaining the genotype data of all snakeheads on the SNP1 to SNP50000 markers; Cluster analysis was performed based on the genotype data to analyze the genetic structure of the snakehead.

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