Grass carp 50K whole genome SNP chip and application thereof

Abstract

The invention discloses a grass carp high-density whole genome 50K SNP (Single Nucleotide Polymorphism) chip and application thereof. Genetic typing objects of the chip comprise 49 and 635 SNP sites on the whole genome of the grass carp, the SNP sites cover 23835 genes of the grass carp, and the positions and genotypes of the SNP sites on the genome (PRJNA1368393) of the grass carp are shown in the specification table 1. The SNP is derived from re-sequencing results of 351 samples, is rich in gene region sites, high in typing accuracy and high in repeatability, is an accurate and efficient molecular breeding chip, can be widely applied to application scenes of grass carp germplasm resource identification, genetic diversity evaluation, target character gene mining and whole genome selective breeding, and has a wide application prospect. The method has the advantage of low detection cost and is beneficial to further improving the grass carp breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of gene chip technology, specifically relating to a grass carp 50K whole genome SNP chip and its application. Background Technology

[0002] Grass carp is the top-selling of my country's "four major freshwater fish" and the world's highest-yielding single farmed fish species. For many years, its production has consistently ranked first among my country's freshwater aquaculture products. However, the industry currently faces severe challenges such as germplasm degradation and disease threats. Genetic selection can significantly improve grass carp's growth rate, disease resistance, feed conversion rate, and survival rate, directly enhancing economic benefits. However, traditional breeding methods are time-consuming, and environmental factors have a significant impact on phenotypic traits, limiting their accuracy.

[0003] Molecular marker technology can rapidly and accurately assess the genetic diversity of these germplasm resources, and through marker-assisted selection, it can be precisely and efficiently applied to existing aquaculture strains to achieve a "strong combination." Among these technologies, SNP microarray technology involves immobilizing SNP markers on a vector to form a dense array of oligonucleotide probes, which then react with target DNA in an allele-specific manner. The presence and strength of the resulting signal determine the polymorphism of the SNP locus. This technology allows for rapid, high-density scanning of the entire crop genome, and is particularly useful for genotyping large population samples in breeding work. The cost per detection locus is very low, making it a highly integrated, high-throughput, miniaturized, and automated method for SNP detection.

[0004] Genome-wide SNP genotyping is a core technique for analyzing the association between genetic variations and traits across the entire genome. Medium-low density and medium-high density SNP / INDEL genotyping technologies, based on pinpoint sequencing of captured targets (cGPS), effectively combine the advantages of high flexibility in site selection, sample quantity flexibility, high throughput, high sensitivity, and low cost, enabling high-throughput targeted screening of specific gene loci variations across the entire genome. SNP liquid-phase chips developed based on molecular markers and cGPS are genotyping tools specifically designed for detecting SNP loci. They use designed specific probes to capture and enrich SNP loci in target regions, and then combine this with high-throughput sequencing technology for genotyping.

[0005] Currently, SNP chips have been developed for several aquaculture species, including large yellow croaker and snakehead, and 20K liquid-phase chips for grass carp are also available. However, the SNP loci enriched in a single chip cannot be associated with all breeding traits. As a major freshwater aquaculture species in my country, grass carp's reference genome data is constantly being updated, making it urgent to develop more accurate and higher-density SNP chips for the selection of superior traits. Summary of the Invention

[0006] The purpose of this invention is to provide a high-density whole-genome chip for grass carp, named "Grass Carp 50K Whole Genome SNP Chip". This chip has advantages such as high throughput, short cycle time, and low cost, and can be used for grass carp germplasm resource identification and genetic diversity assessment, discovery of genes for important growth and stress resistance traits, and whole-genome selection breeding.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] In a first aspect, this invention provides a grass carp whole-genome SNP chip. Probe-designable regions were scanned in the grass carp genome, and then, using resequencing data from 351 grass carp samples, a total of 9,491,990 original variant sites were identified. Then, 103 sites were sampled from the 351 original sample populations, and the heterozygosity distribution of SNPs in the two populations was statistically analyzed, resulting in a set of 2,949,432 SNPs with high heterozygosity and stability. Of these, 2,405,574 sites were found in the target region of grass carp, and after further screening, 49,635 SNPs were obtained, covering 23,835 genes. The locations and genotypes of these SNPs on the grass carp reference genome (PRJNA1368393) are shown in Table 1. Probes were synthesized based on the 49,635 screened SNP sites to fabricate a grass carp 50K whole-genome SNP chip.

[0009] In a second aspect, this invention provides the application of the aforementioned grass carp 50K whole-genome SNP chip. This invention provides a set of probes for detecting combinations of SNP loci in the grass carp whole genome, used to capture 49,635 loci provided by this invention. These probes can be used for grass carp germplasm resource identification and genetic diversity assessment, target trait gene discovery, and whole-genome selection breeding, etc.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] Compared with similar grass carp 20K chips, this invention has more samples from probe sources, more accurate reference genomes, stricter screening criteria, higher genome coverage, and can more effectively identify functional genes and analyze the genetic background of breeding materials.

[0012] Genotyping verification of grass carp samples showed that the 50K whole-genome SNP chip for grass carp has high genotyping accuracy, good stability, and strong repeatability. It can be used for SNP genotyping screening and detection of grass carp candidate parents and offspring, providing reliable support for related breeding work and promoting the development of aquatic seed industry. Attached Figure Description

[0013] Figure 1 Example 1: Flowchart of grass carp SNP site screening.

[0014] Figure 2 Distribution map of the 49,635 grass carp SNP loci screened in Example 1 on the chromosome.

[0015] Figure 3 The genotype detection rate of SNP marker sites in the experimental samples in Example 2.

[0016] Figure 4 Example 3: PCA diagram of kinship analysis of grass carp population.

[0017] Figure 5 Manhattan scatter plot of genome-wide association analysis of grass carp resistance to GCRV virus in Example 4. Detailed Implementation

[0018] Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art. The invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0019] Example 1: Design and fabrication of a 50K whole-genome SNP chip for grass carp

[0020] The SNP collection screening process is as follows: Figure 1 As shown, the specific steps are as follows:

[0021] 1. Genome Resequencing: 351 grass carp samples from different water sources were collected, including 165 samples from the Yangtze River main stream, 67 from the Xiangjiang River system, 65 from the Pearl River system, and 68 samples from unknown water systems. These samples are highly representative of grass carp samples in my country. DNA was extracted and libraries constructed, and after quality control, the samples were sequenced. The raw sequencing data were filtered for quality control using the software FastP (v0.23.1).

[0022] 2. Sequencing Data Alignment: Bowtie2 (v2.1.0) software was used to align the quality-controlled clean reads to the grass carp reference genome (accession number: PRJNA1368393) uploaded to the NCBI database. The genomic location of each read was determined, and the reads were sorted according to their alignment positions to generate a SAM format result file. Picard (v2.20.7) software was used to remove duplicate reads to improve alignment quality. A total of 9,491,990 original variant sites were identified.

[0023] 3. Sequencing Data Variance Detection and Annotation: Variance detection for individual samples was performed using HaplotypeCaller in GATK (v4.1.8.1) software. Then, CombineGVCFs and GenotypeGVCFs were used to merge the gvcf results for all samples. Next, Picard and GATK were used to label and filter VCF files containing all samples, with the following filtering criteria: QD < 2.0 || FS > 60.0 || MQ < 35.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0 || SOR > 3.0. VCF files containing only SNP variant sites were then selected.

[0024] 4. The heterozygosity distribution of the SNPs in the original sample population and the sampling population were statistically analyzed. The heterozygosity mean and standard deviation of the SNPs in the two populations were statistically analyzed. 2,949,432 loci with a mean ≥0.1 and a heterozygosity standard deviation ≤0.01 were screened.

[0025] 5. 34,555 genes from the reference genome were selected as target regions for testing. 2,405,574 sites falling within 10K of these regions and their flanking sequences were screened, covering 28,278 genes (including 26,196 neighboring genes). Among these, 1,658,110 SNPs fell onto genes, encompassing 22,427 genes.

[0026] 6. The candidate SNP set for the target region was sorted by heterozygosity. For each gene, the SNP with the highest heterozygosity was selected first. The remaining markers were further screened according to the criteria of preferentially falling in the gene region and the inter-marker region above 2K, resulting in 72,211 SNPs covering 24,601 genes.

[0027] 7. Using the aforementioned 72,211 SNP loci, a liquid-phase microarray was fabricated, and 116,972 probes were synthesized. This microarray was used to detect 35 grass carp samples from other sources. Loci with a marker detection rate of over 70% in each individual were selected, ultimately yielding 49,635 SNPs covering 23,835 genes. Of these, 35,536 SNPs were located on genes, and 14,099 SNPs were located in upstream or downstream regions of genes. There were 10,514 genes with two SNP loci, 7,643 genes with three SNP loci, and 5,678 genes with only one SNP locus. The distribution of SNP loci on the chromosome is shown below. Figure 2 As shown in Table 1, the SNP locus information is expressed in the form of chromosome: physical location_reference genotype / variant allele.

[0028] Table 1 Information on 49,635 SNP sites

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[0104] Example 2: Evaluation of the genotyping effect of the 50K whole-genome SNP chip in grass carp

[0105] A 50K whole-genome SNP chip for grass carp was designed and fabricated using the 49,635 SNP loci screened in Example 1, and genotyping was performed on 35 grass carp samples. Sequencing and data analysis confirmed an average detection rate of 97.46 ± 0.87% for the 49,635 loci. Additionally, more than two replicate sequencing runs were performed on 7 samples, with a SNP genotyping concordance rate of 99.49 ± 0.11% for the same samples. The specific steps are as follows:

[0106] 1. DNA Extraction and Adapter Ligation: DNA was extracted from the samples using a genomic DNA extraction kit. After quantitative quality testing, the DNA was randomly fragmented using the YZSeq™ Tn5 library preparation kit to a fragment size of 300-400 bp. Subsequently, the fragmented DNA was ligated to sequencing adapters using ligase. Sequencing primers with barcodes were added, and PCR amplification was performed using a high-fidelity PCR reaction system.

[0107] 2. Library hybridization and capture: Fragmented DNA linked to sequencing adapters is hybridized with biotinylated probes targeting the genome. The biotinylated hybridization products are enriched using streptavidin-coated magnetic beads.

[0108] 3. Library elution and PCR enrichment: The eluted and enriched libraries were then accurately measured using real-time quantitative PCR to ensure library quality.

[0109] 4. Sequencing and Data Filtering: After the library passes quality checks, sequencing is performed. The raw sequencing data is quality controlled using the software FastP. The main steps include: (1) removing adapter sequences from the reads; (2) removing low-quality bases at the 5' and 3' ends; (3) setting the minimum length of the reads and removing reads with a length < 40; (4) removing unqualified bases with a base quality value < 20.

[0110] 5. Sequencing data alignment: Sequence alignment compares the similarity of the sequencing data of the experimental samples with known reference genome data to determine the genomic location of each read, and generates an alignment result file containing the alignment information of each sequencing sequence on the genome.

[0111] 6. Sequencing Data Variation Detection and Annotation: HaplotypeCaller was used for variation detection in individual samples. Then, CombineGVCFs and GenotypeGVCFs were used to merge the gvcf results of all samples. Picard and GATK software were used to label and filter VCF files containing all samples, selecting VCF files containing only SNP variant sites. Finally, the genotyping results of 35 grass carp samples were obtained. The results are as follows: Figure 3 As shown, the grass carp 50K whole genome SNP chip demonstrated high target locus detection rate, good stability, and accurate and reliable genotyping results when genotyping 35 grass carp samples. It can be fully used for SNP genotyping detection of different grass carp samples.

[0112] Example 3: Application of Grass Carp 50K Whole Genome SNP Array in Phylogenetic Identification Analysis

[0113] Genetic analysis was performed on 211 grass carp using a 50K whole-genome SNP chip. The genotyping results were quality controlled using PLINK software, and phylogenetic relationships were identified. The results showed that the analytical results were consistent with the actual grouping results. Figure 4 This chip can be used for germplasm resource evaluation.

[0114] Example 4: Application of grass carp 50K whole-genome SNP array in genome-wide association analysis of disease resistance traits

[0115] Sixty-one 3-month-old grass carp samples were collected and subjected to a type II GCRV virus infection experiment. Disease resistance (survival) and susceptibility (mortality) data were collected, 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 a sample deletion rate of 10%, resulting in 42,865 high-quality SNP loci. Subsequently, PLINK's Fisher's exact test was used to perform genome-wide association analysis (GWAS) on the anti-GCRV virus trait. Ultimately, 11 SNP loci associated with this trait were located, distributed on chromosomes 6 and 21. Figure 5 Through gene annotation, genes related to transcription factors, transmembrane proteins, and cytoplasmic regulatory factors of innate immunity, which are closely related to the immune system, were successfully annotated. Therefore, the 50K whole-genome SNP chip described in this invention can be used to genotype grass carp and obtain accurate genome-wide association analysis results, which can be used for the breeding of disease-resistant grass carp.

[0116] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A molecular probe assembly, characterized in that, The molecular probe combination was used to detect the 49,635 SNP sites shown in Table 1 of the instruction manual.

2. The application of the molecular probe combination described in claim 1 in the preparation of grass carp whole genome SNP chip.

3. A high-density whole-genome 50K SNP chip for grass carp, characterized in that, The chip is loaded with the molecular probe assembly as described in claim 1.

4. The application of the molecular probe combination described in claim 1 in the identification of grass carp germplasm resources or the assessment of genetic diversity.

5. The application of the molecular probe combination of claim 1 in the analysis of target traits or gene localization of grass carp.

6. The application of the molecular probe combination described in claim 1 in grass carp breeding.

7. The application of the SNP chip described in claim 3 in the identification of grass carp germplasm resources or the assessment of genetic diversity.

8. The application of the SNP chip according to claim 3 in the analysis of target traits or gene localization of grass carp.

9. The application of the SNP chip as described in claim 3 in grass carp breeding.

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