SNP (Single Nucleotide Polymorphism) liquid phase breeding chip for grass carp and application thereof

Abstract

The invention belongs to the technical field of molecular detection, and particularly discloses a grass carp SNP (Single Nucleotide Polymorphism) liquid phase breeding chip and application thereof. The invention provides a grass carp SNP liquid phase breeding chip, a genetic typing object of the chip comprises 21546 SNP sites on a grass carp whole genome, the chip is high in site polymorphism, rich in functional sites and gene region sites and contains various detection markers, the sites are uniformly distributed on chromosomes, and the chip is an accurate and efficient molecular breeding chip. The chip can be widely applied to different application scenes such as grass carp germplasm resource identification and genetic diversity evaluation, genetic map construction and QTL positioning, whole genome association analysis, mining identification and function analysis of important character genes, whole genome selective breeding and the like, and is beneficial to improving the grass carp breeding efficiency; a new variety with excellent characters such as high growth speed is cultivated, and healthy and sustainable development of the grass carp breeding industry is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection, and particularly relates to a grass carp SNP liquid breeding chip and its application. Background Art

[0002] At present, the sustainable development of grass carp aquaculture faces many challenges, such as germplasm degradation and the outbreak of various infectious diseases, which bring incalculable economic losses to the development of the aquaculture industry in China and even the world. Therefore, accelerating the cultivation of grass carp towards high quality is very important for the development of the grass carp industry in China, and molecular breeding markers are one of the means to promote this goal.

[0003] The SNP chip is a high-throughput genotyping technology used to detect variations at single nucleotide polymorphism (SNP) sites in the genome and is one of the important technical means for molecular breeding. SNP refers to the polymorphism caused by the mutation of a single nucleotide (such as transition, transversion, insertion or deletion) in the genomic DNA sequence. This technology quickly and accurately detects multiple SNP sites by hybridizing a large number of specific probes with the target DNA.

[0004] Whole-genome SNP genotyping is the core technical means for analyzing the association between gene genetic variations and traits across the whole genome. Medium-low density and medium-high density SNP / INDEL genotyping technologies, based on the multiplex PCR pinpoint sequencing genotyping technology (Genotyping by Pinpoint Sequencing of multiplex PCR products, abbreviated as mGPS) or the pinpoint sequencing genotyping technology based on liquid-phase capture of target region genomic sequences (Genotyping by Pinpoint Sequencing of captured targets, cGPS), effectively combine the advantages of high flexibility in site selection, flexibility in the number of samples, high throughput, high sensitivity, and low cost, and can achieve high-throughput targeted screening of specific gene locus variations across the whole genome. The SNP liquid chip developed based on molecular markers and cGPS is a genotyping tool specifically used to detect SNP sites. It captures and enriches the SNP sites in the target region by designing specific probes, and then combines high-throughput sequencing technology for genotyping. Currently, there is no research on applying the SNP liquid chip to grass carp genetic breeding. Summary of the Invention

[0005] To transform the research results of the grass carp functional genome and better serve the grass carp breeding work, target regions with uniform distribution of 21K on chromosomes were selected. The present invention developed, verified and evaluated a high-throughput 21K liquid chip, named "Grass Carp 21K Liquid Chip". This chip has an extremely high capture rate and genotyping accuracy, and can be used as a fast and universal genotyping tool for future grass carp genome breeding and research.

[0006] The specific technical solutions adopted in the present invention are as follows:

[0007] In the first aspect, the present invention provides a combination of grass carp SNP molecular markers, including 21,546 SNP loci on the whole genome of grass carp, and the information of the 21,546 SNP loci is shown in Table 1 of the specification.

[0008] In the second aspect, the present invention provides a grass carp SNP liquid breeding chip, the genotyping object of which is the above-mentioned combination of grass carp SNP molecular markers, and the chip includes probes for detecting 21,546 SNP loci on the whole genome of grass carp.

[0009] Based on the whole genome data of 8 grass carp populations including 80 individuals, highly reliable SNP locus information was obtained by analyzing the sequencing data for subsequent screening. According to indicators such as the chromosomal position, minor allele frequency, missing rate, and heterozygosity rate of SNP loci, 8,585,974 SNP loci with high quality, high polymorphism, and uniform chromosomal distribution were screened. During the locus screening process, gene region loci were preferably selected, and the gene coverage rate was as high as over 95%. 21,546 SNP loci with high polymorphism and good universality for chip development were obtained, and probes were designed for the 21,546 loci to obtain the grass carp 21K SNP liquid breeding chip.

[0010] In the third aspect, the present invention provides the application of the above-mentioned grass carp SNP liquid breeding chip in aspects such as grass carp germplasm resource identification and genetic diversity assessment, genetic map construction and QTL mapping, genome-wide association analysis, mining and identification and functional analysis of genes for important traits, and genome-wide selection breeding.

[0011] Furthermore, the method of the application is to genotype grass carp samples using the grass carp SNP liquid breeding chip.

[0012] The present invention has the following beneficial effects:

[0013] 1. The 21K SNP liquid breeding chip for grass carp is based on the whole-genome data of 8 grass carp populations including 80 individuals. By preferentially selecting gene region loci, the gene coverage rate is over 95%. 21,546 SNP loci with high quality, high polymorphism and uniform distribution on chromosomes are screened, and a precise and efficient genotyping identification chip with probes for 21,546 molecular markers is obtained.

[0014] 2. Gene typing verification is carried out on grass carp samples. After verification, the overall performance of the 21K SNP liquid breeding chip for grass carp is excellent: high genotype detection rate, high genotype accuracy and good stability, and it can be fully used for SNP typing detection of different grass carp samples.

[0015] 3. The present invention can be widely used in different application scenarios such as germplasm resource identification and genetic diversity assessment, genetic map construction and QTL mapping, genome-wide association analysis, mining and identification and functional analysis of genes for important traits, and genome-wide selection breeding of different grass carp samples, which is of great significance for improving the breeding efficiency of grass carp species and promoting the high-quality development of China's grass carp industry.

[0016] 4. The precise positioning sequencing typing technology based on liquid-phase capture of genomic sequences in the target interval can not only type the target loci, but also detect all genetic variation loci in the target interval and a certain range around it, and the output result has a large amount of information. Based on high-throughput sequencing technology, this detection method has a high detection throughput, a large amount of data produced at one time, and can cover the detection of nearly a thousand materials at the same time; it is also applicable to mainstream second-generation sequencing platforms such as illumina and MGI, and has wide platform applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : The screening process of grass carp SNP loci in Example 1.

[0018] Figure 2 : The distribution map of 21,546 grass carp SNP loci screened in Example 1 on chromosomes.

[0019] Figure 3 : The genotype detection rate of SNP locus marker sites of experimental samples in Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0021] Example 1: Design and Preparation of the 21K SNP Liquid Breeding Chip for Grass Carp

[0022] The genomic resequencing data and VCF results of 8 grass carp populations including 80 individuals are used as the set of SNP sources. The SNP set screening process is as Figure 1As shown below, the specific steps are as follows:

[0023] Step 1: The minor allele frequency (MAF) ≥ 0.2, the deletion rate ≤ 0.1, the heterozygosity rate ≤ 0.3, the average depth ≥ 10, and all candidate sites meet the selection criteria.

[0024] Step 2: Refer to the genome of grass carp, screen and generate probes with a target region larger than 122k; determine that the number of SNPs within each 100bp window is greater than 1, the GC content is 20%-80%, and there are neither hairpin structures nor dimers; finally, 8,585,974 SNPs in 101,133 target regions meet the probe design requirements.

[0025] Step 3: Retain the head and tail sites of the chromosome to ensure the coverage of the sites on the reference genome. At the same time, the site density is adjusted according to the physical distance. The physical distance between adjacent SNPs is set to 40kb, so that all sites are evenly distributed on the reference genome. As a result, 20,290 sites are retained.

[0026] Step 4: Synthesize a probe set that matches 21K target sites, and then test it on 12 grass carps. Construct sequencing libraries for 12 samples, perform hybridization capture, and conduct high-throughput sequencing after strict quality control of the libraries. The raw data obtained needs to be processed such as filtering. Reads with adapter contamination and low quality are removed. Finally, the high-quality clean reads are aligned with the reference genome of grass carp using the BWA software, and then the GATK software is used to analyze the variant sites of the sequencing results to obtain the genotype data of 12 test samples. The average detection rate of 12 samples is 97.6%, and the genotype consistency rate of repeated samples is 98.7%. According to the results of gene typing, 325 sites with low detection rates are optimized and removed, and 1,581 sites with high polymorphism and uniform distribution are added. A total of 21,546 SNP sites and 23,335 probes designed for these sites are obtained.

[0027] The 21,546 SNP sites screened are evenly distributed on 24 chromosomes of grass carp. The site distribution map is as Figure 2 shown, and the specific information is shown in Table 1 below. The SNP site information is represented in the form of chromosome: physical position: reference genotype: variant allele genotype.

[0028] Table 1 Information of 21,546 SNP sites (CHROM:POS:ref:alt)

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[0100] Functional annotation of 21,546 SNP loci showed that 6,590 SNP loci (30.56%) were located within gene regions, including exons, introns, 3'UTRs, and 5'UTRs. The two largest categories within gene regions were introns (4,519 SNP loci) and exons (1,599 SNP loci). In contrast, the distribution of non-gene SNP regions was as follows: 3,507 SNP loci (16.28%) were upstream (within 1 kb of the start codon), 2,573 SNP loci (11.94%) were downstream (within 1 kb of the stop codon), and 8,876 SNPs (41.20% of the region) were intergenic. This distribution ensures that the array simultaneously captures regulatory and coding regions, thus facilitating the identification of functional genetic variants associated with key traits.

[0101] For the 21,546 SNP loci screened, a 21K liquid breeding chip for grass carp was developed using the targeted sequencing genotyping technology (cGPS technology). The principle is to design probes for the target interval sequence based on an optimized thermodynamic stability algorithm model, and use synthetic specific probes to perform liquid hybridization capture and enrichment on multiple different target sequences located at different genomic positions. Then, a sequencing library is constructed for the captured and enriched target genomic sequence, and high-throughput sequencing is carried out to obtain the genotypes of all SNP / InDel loci within the target region.

[0102] This technology involves the following steps:

[0103] (1) Extraction and quality control of genomic DNA

[0104] The magnetic bead method is used to extract the DNA of the sample, and the quality of the DNA sample is detected. The quality detection includes measuring the DNA concentration with a Qubit fluorometer and detecting the integrity of the DNA by 1% agarose gel electrophoresis. The samples that pass the quality inspection are used for library preparation.

[0105] (2) Construction and quality control of the GPS library

[0106] a. Use a fragmentation enzyme to digest the DNA sample, repair the ends of the digestion, add an A base at the 3' end, and detect the fragment size by agarose gel electrophoresis. b. Use T4 ligase to ligate the sequencing adapter to the DNA fragment, and purify the ligation product using magnetic beads. The purified product is detected for concentration with a Qubit fluorometer and the fragment size is detected by agarose gel electrophoresis. c. Perform PCR amplification on the purified ligation product, and use magnetic beads to screen the amplified product for fragments. The product after fragment screening is detected for concentration with a Qubit fluorometer and the fragment size is detected by agarose gel electrophoresis. d. Take 200 ng of the completed library, add the probe and hybridization reagent, and incubate at 50 °C for 16 - 24 hours to complete the hybridization reaction. Use magnetic beads for target segment capture, wash the captured product with the washing solution to remove non-specific binding fragments, and then perform another round of PCR amplification. Detect the library concentration with a Qubit fluorometer and the fragment size by agarose gel electrophoresis. After the concentration and fragment size are determined to be qualified, the construction of the cGPS sequencing library is completed. The prepared library is subjected to high-throughput sequencing using a BGI sequencer, and the sequencing strategy is PE150.

[0107] (3) Data analysis

[0108] The original data after high-throughput sequencing is subjected to quality control filtering and other processes. The FASTP software is used to remove adapter fragments and low-quality reads to obtain high-quality Clean Reads. The obtained Clean Reads are aligned with the reference genome using the BWA software, and position sorting is performed to obtain the sorted bam file of the sample. The GATK software is used to analyze the variant sites of the sequencing results to obtain the original vcf variant result file. Judgments are made based on the proportion of the number of supporting Reads of different Alles at the target site. When the support ratio of mutant reads is ≥0.8 or ≤0.2, the site is judged to be a homozygous genotype. When the support ratio of mutant reads is between 0.2 and 0.8, it is judged to be a heterozygous genotype. An internally written perl script is used to process the original vcf variant result file to obtain the converted vcf variant result file. Finally, the genotyping results of each target SNP in a specific individual are obtained through high throughput, realizing high-throughput SNP genotyping.

[0109] Example 2: Evaluation of the Genotyping Effect of the Grass Carp SNP Liquid Breeding Chip

[0110] To verify the genotyping effect of the grass carp 21K SNP liquid breeding chip, the grass carp 21K SNP liquid breeding chip designed in Example 1 was used to perform genotyping detection on 103 grass carp samples. After sequencing and data analysis, the site detection rate of the 103 grass carp species samples was between 98.28% and 99.24% (as Figure 3 shown), and the average detection rate was 98.89%. For the comparison of the genotype results of 6 replicated samples, the average consistency rate of the genotypes of the technical replicated samples was 99.40% (in the consistency comparison, if one of the two results at a certain site is missing, this site is not included in the consistency statistics). The results show that when the grass carp SNP liquid breeding chip performs genotyping on the test materials, the target site detection rate is high, the stability is good, the genotyping results are accurate and reliable, and it can be fully used for the SNP genotyping detection of different grass carp samples.

[0111] This specific implementation manner is only an interpretation of the present invention and does not limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A grass carp SNP molecular marker combination, characterized in that: It includes 21546 SNP sites on the whole genome of grass carp, and the information of the SNP sites is shown in Table 1 of the specification.

2. A grass carp SNP liquid phase breeding chip, characterized in that: The genotyping object of the chip is the grass carp SNP molecular marker combination described in claim 1.

3. Application of the grass carp SNP liquid phase breeding chip according to claim 2 in the identification of grass carp germplasm resources and genetic diversity assessment, genetic map construction and QTL positioning, whole genome association analysis, mining and identification of important trait genes and functional analysis, and whole genome selection breeding.

4. The use according to claim 3, characterized in that: The application method is to use the grass carp SNP liquid phase breeding chip to perform genotyping on grass carp samples.

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