Silver carp 20K liquid phase chip and application thereof

Abstract

The invention discloses a silver carp 20K liquid phase chip and application thereof, the liquid phase chip comprises a probe combination of 20,909 SNP molecular markers covering a whole genome, the physical positions of the 20,909 SNP molecular markers are determined based on sequence alignment of the silver carp genome (GCA041475455.1), and the site information of the 20,909 SNP molecular markers is shown in the specification table 1. The invention provides a silver carp first type whole genome SNP liquid phase breeding chip, which effectively reduces the application cost of silver carp in genetic diversity analysis, QTL positioning, GWAS analysis and the like in scientific research, solves the problem that no applicable product exists in silver carp molecular auxiliary breeding and whole genome selective breeding, accelerates the progress of silver carp basic research and breeding, and has a wide application prospect. The method is of great significance in improving the utilization efficiency of silver carp germplasm resources, accelerating the breeding process of excellent characters and improving the economic value and environmental adaptability of bred varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of gene chips, and particularly relates to a silver carp 20K liquid chip and its application. Background Art

[0002] In the field of aquaculture, the silver carp is one of the important freshwater cultured fish in China, and its output occupies a significant position in the fishery economy. However, for a long time, the traditional selection and breeding of silver carp mainly rely on phenotypic characteristics, which is inefficient and limited in accuracy, and it is difficult to meet the urgent needs of modern fishery for high-quality and high-yield silver carp varieties. With the rapid development of molecular biology technology, SNP molecular markers have gradually become a powerful tool for fish genetic breeding research due to their many advantages such as large quantity, wide distribution, high stability, and easy detection. By accurately identifying and analyzing SNP loci within the whole genome of silver carp, it is possible to deeply understand its genetic structure and variation rules, providing a key genetic information basis for molecular marker-assisted breeding of silver carp, thus greatly accelerating the breeding process of excellent silver carp varieties.

[0003] Although SNP molecular markers theoretically provide broad prospects for silver carp genetic research, in practical applications, traditional SNP detection methods such as PCR-RFLP and direct sequencing have disadvantages such as low throughput, high cost, and cumbersome operation, and it is difficult to achieve efficient and accurate detection of a large number of samples and numerous SNP loci. As an emerging high-throughput genotyping platform, liquid chip technology can immobilize a large number of SNP probes on carriers such as microspheres and perform hybridization reactions with the DNA samples to be detected in a liquid phase environment. With its characteristics of high throughput, high accuracy, and strong flexibility, it brings new opportunities to solve the SNP detection problem. Applying liquid chip technology to silver carp SNP molecular marker detection is expected to break through the traditional technical bottleneck and achieve large-scale and precise analysis of silver carp genetic information.

[0004] Currently, the aquaculture industry is facing severe challenges such as increased resource and environmental constraints and increasingly fierce market competition, and there is an urgent need to enhance industrial competitiveness through scientific and technological innovation. For silver carp aquaculture, cultivating new varieties with comprehensive excellent traits such as fast growth rate, strong disease resistance, and good meat quality is the core task for achieving sustainable industrial development. By means of SNP molecular markers and liquid chip technology, it is possible to accurately screen genetic markers closely associated with these excellent traits and then carry out precise molecular design breeding. This not only helps to improve the economic and social benefits of silver carp aquaculture, but also has profound strategic significance for ensuring the stable supply and sustainable development of China's freshwater fishery. Summary of the Invention

[0005] The main objective of the present invention is to propose a silver carp 20K liquid-phase chip and its applications, aiming to provide a silver carp molecular marker chip with high throughput, high accuracy, low cost, multiple functions, and a wide range of application scenarios, which can be widely used in aspects such as genome-wide association analysis, germplasm resource identification, genetic diversity assessment, genetic map construction and QTL mapping, molecular marker-assisted selection, and genome-wide selection of silver carp.

[0006] To achieve the above objective, the present invention proposes a silver carp 20K liquid-phase chip. The liquid-phase chip includes a probe combination of 20,909 SNP molecular markers covering the entire genome. The physical positions of the 20,909 SNP molecular markers are determined based on sequence alignment with the silver carp genome (GCA_041475455.1), and the locus information of the 20,909 SNP molecular markers is shown in Table 1 of the specification.

[0007] Table 1 Locus information of 20,909 SNP molecular markers

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[0061] Preferably, 459 of the molecular markers are associated with the economic traits of silver carp, and the economic traits are body weight and body height related to growth traits, oxygen consumption rate related to hypoxia tolerance traits, and sex traits; at the same time, the molecular markers also contain 460 population-specific loci that can distinguish different varieties of silver carp.

[0062] The present invention also provides a kit for detecting a combination of silver carp SNP molecular markers, including the liquid-phase chip as described above.

[0063] The present invention also provides an application of the silver carp 20K liquid-phase chip as described above or the kit as described above in the detection of silver carp DNA samples.

[0064] Preferably, the application includes the application in genotyping of silver carp samples.

[0065] Preferably, the application includes the application in analyzing the genetic relationship between silver carp and its related species.

[0066] Preferably, the application further includes the application in analyzing the trait association of silver carp breeding materials.

[0067] The present invention also provides a method for preparing a silver carp whole-genome liquid-phase breeding chip, comprising the following steps:

[0068] (1) Screening out high-quality loci from the silver carp resequencing data, and preferably retaining loci with high MAF values based on the principle of uniform distribution;

[0069] (2) Screen SNP loci associated with growth, hypoxia tolerance, and sex traits from the silver carp whole-genome association analysis data, and simultaneously obtain specific loci that can distinguish different varieties and the silver carp population in Jianli, Hubei from the results of allele frequency difference analysis of the silver carp resequencing data;

[0070] (3) Remove the loci that cannot design probes on both sides of the locus from the above loci, and remove the probes that cannot be uniquely aligned on the genome and contain repetitive sequences in the flanking sequences;

[0071] (4) Design probes for the above SNP locus set that meet the standards, construct a probe pool, and obtain a silver carp whole-genome liquid breeding chip.

[0072] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0073] (1) The present invention provides a first silver carp whole-genome SNP liquid breeding chip, which effectively reduces the costs of genetic diversity analysis, QTL mapping, GWAS analysis, etc. in silver carp research, solves the problem of no applicable products for silver carp molecular-assisted breeding and whole-genome selection breeding, accelerates the process of silver carp basic research and breeding, and is of great significance for improving the utilization efficiency of silver carp germplasm resources, accelerating the breeding process of excellent traits, and enhancing the economic value and environmental adaptability of cultured varieties.

[0074] (2) Based on the data of 624 silver carp germplasm resources with rich diversity collected nationwide and the sequencing data of 310 selected breeding populations, the present invention optimizes 20,909 molecular marker loci with strong representativeness, high polymorphism (MAF mean 0.31), good universality, high coverage rate on the genome (average coverage rate 99.46%), and uniform distribution; this chip has a high detection throughput, a large amount of output result information, a high detection rate of target loci, and accurate and reliable genotyping results (repetitive consistency rate 99.20%); this chip has the characteristic of strong universality, and the average detection rate of the present invention in silver carp can reach 99.03%, and the average detection rate in bighead carp can reach 91.87%, which can meet the detection needs of silver carp and bighead carp.

[0075] (3) The liquid chip involved in the present invention adds 459 functional markers related to traits such as silver carp growth, sex, hypoxia tolerance, and oxygen consumption rate, which is beneficial for molecular marker-assisted selection, directional improvement, multi-gene pyramiding breeding, and the mining, identification, and functional analysis of genes for important traits; at the same time, 460 SNP fingerprint map markers of specific varieties are added to achieve rapid and accurate identification of silver carp varieties, which has great application value in the process of silver carp variety improvement and variety breeding.

[0076] (4) Compared with traditional solid-phase chips, the liquid-phase chip involved in the present invention has higher flexibility and can add labeling sites at any time according to the needs of the application scenario. At the same time, relying on the next-generation sequencing platform, the liquid-phase chip has a lower genotyping cost, a high detection throughput, a large amount of data output at one time, and can cover the detection of nearly a thousand materials at the same time, providing a technical means for large-scale genotyping. Based on the targeted capture sequencing technology, the liquid-phase chip can not only genotype the target sites, but also accurately genotype the genetic variation sites within a certain range around the target sites. Therefore, more SNP genotyping information than the expected labeling sites can be obtained, providing richer site information support for silver carp molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 It is a distribution map of the silver carp 20K SNP liquid breeding chip sites on chromosomes provided by the present invention;

[0078] Figure 2 It is a MAF distribution map of the silver carp 20K SNP liquid breeding chip sites provided by the present invention;

[0079] Figure 3 It is the genotype detection rate of the silver carp 20K SNP liquid breeding chip sites in the samples provided by the present invention;

[0080] Figure 4 It is a map of the consistency rate of genotypes of repeated samples of the silver carp 20K SNP liquid breeding chip provided by the present invention;

[0081] Figure 5 It is a phylogenetic tree constructed based on the genotyping information of bighead carp and different silver carp populations using the silver carp 20K SNP liquid breeding chip provided by the present invention;

[0082] Figure 6 It is a QQ plot and a Manhattan plot of the genome-wide association analysis for verifying the low-oxygen tolerance trait of silver carp using the silver carp 20K SNP liquid breeding chip provided by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0083] 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. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not indicated by the manufacturer are all conventional products that can be obtained through commercial purchase. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0084] Example 1 Design and Preparation of Silver Carp Whole-Genome SNP Liquid Breeding Chip

[0085] 1. Collection of Diverse Silver Carp Materials

[0086] To obtain a rich and diverse set of whole-genome SNP sites, 624 silver carp germplasm resources from different regions such as Hubei, Hunan, Shaanxi, Inner Mongolia, Hebei, Jiangsu, and Zhejiang, as well as 310 samples from different selected populations (silver carp families, Changfeng silver carp, Changfeng silver carp No. 2, and common silver carp) were collected. The specific information is shown in Table 2. Whole-genome resequencing was performed on the diverse samples from the above different sources.

[0087] Table 2 Information on silver carp resequencing samples

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[0090] 2. Whole-genome resequencing of silver carp

[0091] The collected silver carp samples were subjected to DNA extraction and whole-genome resequencing. The specific steps include:

[0092] (1) DNA was extracted from the sample tissues using the magnetic bead method. (2) The DNA samples were enzymatically fragmented using dsDNAFragmentase, the ends of the enzyme digestion were repaired, and an A base was added to the 3' end. (3) The sequencing adapters were ligated to the fragmented DNA, and the ligation products were purified using magnetic beads. (4) The ligation products were amplified by PCR, and the PCR products were screened for fragments using magnetic beads. (5) The linear library was denatured into single strands and then circularized. After digesting the uncircularized linear DNA molecules, a single-stranded circular library was obtained. (6) The single-stranded circular DNA molecules underwent rolling circle replication to form a DNA nanoball (DNB) containing more than 300 copies. (7) The DNB was loaded into the sequencing chip using the loading device MGIDL-T7, and sequencing was performed on the machine through the combined probe anchor polymerization technology. The sequencing strategy was PE150, and the sequencing depth was 10×.

[0093] Sentieon was used to align and detect variants in the resequencing data. The analysis process is as follows:

[0094] (1) Align the reads to the silver carp reference genome (GCA_041475455.1) using Sentieon, sort the positions, and mark the duplicate reads. (2) Detect variant sites for each sample using Sentieon to obtain the variant information for each sample. (3) Perform joint-calling using Sentieon to jointly analyze the gVCFs of all samples and obtain the variant results for each individual in the population. To ensure the accuracy of SNPs, perform preliminary hard filtering on the SNP sites obtained after joint analysis (SNP hard filtering criteria: "QD < 2.0 || FS > 60.0 || MQ < 40.0 || SOR > 3.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0").

[0095] 3. Site Screening

[0096] (1) Genome-wide Sites

[0097] Calculate and statistically analyze the MAF value, detection rate, heterozygosity rate, and sequencing depth of sites from the vcf file containing the SNP variant information of all samples. Screen polymorphic SNP sites with MAF ≥ 0.1, SNP site detection rate ≥ 90%, heterozygosity rate ≤ 40%, and sequencing depth ≥ 10× as initial candidate sites. Use all the above candidate sites for probe design, design probes within 100 bp upstream and downstream of all target sites, with a probe length of about 100 bp and a GC content between 20% - 80%. According to the probe design results, remove probes that cannot be uniquely aligned to the genome and those containing repetitive sequences in the flanking sequences. Based on the principle of uniform distribution, preferentially retain sites with high MAF values, and finally obtain 19,990 genome-wide background sites with high polymorphism.

[0098] (2) Functional Sites

[0099] a. Collect 7 sites related to the growth traits of silver carp and 131 sites related to hypoxia tolerance traits obtained from previous studies.

[0100] b. Collect and organize the sex phenotype data of 105 re-sequenced samples, calculate and statistically analyze the site population metrics in the vcf file containing all variant information of 105 silver carp samples, and filter out sites with a missing rate greater than 0.1, MAF value less than 0.05, and sites located on contigs. Perform genome-wide association analysis using the mixed linear model (MLM), and according to the threshold p = 1e -5 Screen out 210 sites significantly associated with sex.

[0101] c. Collect and organize the growth phenotype data of 97 re-sequenced samples of Changfeng silver carp and 100 re-sequenced samples of common silver carp, and perform genome-wide association analysis using the R package rMVP, including three models: general linear model (GLM), mixed linear model (MLM), and FarmCPU model. Add the kinship matrix and principal components as covariates to the model for correction to reduce the influence of kinship and population structure. According to the threshold line p = 1e -5 , combine the analysis results of the three models, and finally map 111 loci significantly associated with growth traits.

[0102] (3) Population-specific loci

[0103] Extract the genotypes of 108 individuals from a total of 4 populations, namely common silver carp, Changfeng silver carp, new strain Changfeng silver carp No. 2, and Hubei Jianli population, from the above re-sequencing data. First, screen SNPs with a detection rate greater than or equal to 90%, MAF greater than or equal to 0.05, and dimorphic genotypes, and then screen for differential loci between varieties based on allele distribution differences. Finally, 460 variety-specific differentiation loci are obtained.

[0104] After integrating and removing duplicates of all the loci in the above (1)-(3), they jointly constitute the locus set of the silver carp 20K SNP liquid breeding chip, and the final number of loci is 20,909 (Table 1). The average coverage rate of these loci on the silver carp chromosomes is 99.70%, the average interval is 38 Kb, and the 20K loci are evenly distributed on each chromosome, and the density distribution diagram is as Figure 1 shown. The silver carp 20K SNP chip loci have high polymorphism, and the average minor allele frequency (MAF value) is 0.31, and the MAF value distribution is as Figure 2 shown.

[0105] Develop the screened 20,909 SNP loci into a silver carp 20K SNP liquid breeding chip using the liquid-phase probe precise positioning sequencing typing technology (Genotyping by Pinpoint Sequencing of liquid captured target, cGPS) independently developed by Huazhi. cGPS is based on an optimized thermodynamic stability algorithm model to design specific probes for the target interval sequence, and then use the synthesized specific probes to perform liquid-phase hybridization capture and enrichment on multiple different target sequences located at different genomic positions, and then construct a sequencing library and perform high-throughput sequencing on the captured and enriched target genomic sequences to obtain the genotypes of all SNP / InDel loci within the target region.

[0106] Example 2 Application of the silver carp 20K SNP liquid breeding chip in silver carp genotyping

[0107] This embodiment provides a method for genotyping silver carp samples using the 20K SNP liquid breeding chip for silver carp in Application Example 1. The steps are as follows:

[0108] 1. Extraction and quality control of silver carp genomic DNA

[0109] The magnetic bead method is used to extract sample DNA, and the DNA sample is subjected to quality detection. 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.

[0110] 2. cGPS experimental and analysis process

[0111] (1) Take 200 ng of quantitatively qualified genomic DNA, use a fragmenting enzyme to digest the DNA sample, repair the ends of the digestion and add an A base at the 3' end, and detect the fragment size by agarose gel electrophoresis;

[0112] (2) Use T4 ligase to ligate adapter fragments to both ends of the DNA, and use fragment sorting magnetic beads to purify the ligation product. The purified product is detected for concentration with a Qubit fluorometer and the fragment size is detected by agarose gel electrophoresis;

[0113] (3) 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;

[0114] (4) Place the library that passes the quality inspection, the blocking reagent, the RNase inhibitor, and the 20K liquid breeding chip probe on a PCR instrument for hybridization reaction, and incubate at 55 °C for 16 - 24 hours;

[0115] (5) Use streptavidin to capture the hybridization product, amplify and enrich the captured library, and perform PE150 sequencing using the BGI sequencing DNBSEQ-T7 platform;

[0116] (6) The raw data after high-throughput sequencing is processed through quality control filtering, etc. The FASTP software is used to remove adapter fragments and low-quality reads to obtain high-quality Clean Reads. The obtained Cleanreads are aligned with the reference genome using the BWA software, and sorted by position 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 genotype typing results of the target sites.

[0117] Example 3 Evaluation of the genotyping effect of the 20K SNP liquid breeding chip for silver carp

[0118] To verify the genotyping effect of the silver carp 20K SNP liquid breeding chip, the silver carp 20K SNP liquid breeding chip designed and completed in Example 1 was used to perform genotyping detection on 90 silver carp materials (including 13 re-sequencing data samples used for the development of the liquid chip) and 3 bighead carp materials. Five technical replicates were set among the 90 silver carp materials, resulting in a total of 95 silver carp sample data and 3 bighead carp sample data. The specific usage method is as shown in Example 2.

[0119] After sequencing and data analysis, the locus detection rate of the 95 silver carp materials was between 98.50% and 99.57%, and the average detection rate was 99.03%; the locus detection rate of the 3 bighead carp materials was between 91.78% and 91.98%, and the average detection rate was 91.87%. See specifically Figure 3 ; the genotype consistency rate of the technical replicate samples was between 98.90% and 99.40%, and the average consistency rate was 99.21%. See specifically Figure 4 . By comparing the cGPS liquid chip results with the re-sequencing results of 13 re-sequencing samples, the genotype consistency rate was between 95.13% and 96.96%, and the average consistency rate was 95.89%. The above data indicate that when the silver carp 20K SNP liquid breeding chip performs genotyping on the test materials, the target locus detection rate is high, the stability is good, and the genotyping results are accurate and reliable.

[0120] Example 4 Application of the silver carp 20K SNP liquid breeding chip in the analysis of the genetic relationship between silver carp and related species

[0121] The silver carp 20K SNP liquid breeding chip prepared in Example 1 was used to perform genotype identification on 82 silver carp samples and 3 bighead carp samples from different sources (the specific operation method is shown in Example 2). The genotype typing results of 85 samples were extracted, and the genetic distance matrix was calculated using the IBS method of Plink software and cluster analysis was performed to construct a phylogenetic tree. The results showed that the test materials were mainly divided into 6 subgroups, and the classification effect was consistent with the actual classification, as shown in Figure 5 . It shows that the silver carp 20K SNP liquid breeding chip can effectively distinguish silver carp from different sources and can be used to judge the genetic relationship, evolutionary relationship and composition structure between different materials.

[0122] Example 5 Application of the silver carp 20K SNP liquid breeding chip in the analysis of the correlation between traits of silver carp breeding materials

[0123] The 6-month-old Changfeng silver carp in the same breeding population were subjected to hypoxia stress. 107 fish that first showed turning over under hypoxia stress were collected as the hypoxia-sensitive group, and at the same time, 116 fish that turned over last were collected as the hypoxia-tolerant group. The constructed 20K liquid-phase breeding chip of silver carp was used to genotype these 223 Changfeng silver carp (performed according to the method in Example 2). After quality control of the obtained genotyping results and deletion of low-quality loci, 19,257 high-quality SNP loci were screened. Subsequently, the mixed linear model of the Gemma software was used to perform a genome-wide association analysis on the genotype information of these 223 individuals at these loci and the hypoxia tolerance phenotype (sensitive and tolerant binary traits), and the Bonferroni test was used to screen significant loci. Finally, 7 SNP loci significantly associated with the hypoxia tolerance trait of Changfeng silver carp were identified. The QQ plot and Manhattan plot of the genome-wide association analysis of the hypoxia tolerance trait are shown in Figure 6 Figure [0000319]. Annotation of these loci found that 3 SNP loci located on chromosomes 5, 8, and 22 were all located at loci. A total of 32 candidate genes were identified at the 7 loci. Among them, 5 genes such as prdx1, ent1, and nrf2 were differentially expressed in the heart or gill tissues of silver carp under hypoxia stress. These genes are mainly closely related to processes such as stress response, DNA damage repair, immune regulation, and hypoxia-inducible factor regulation. It can be seen that genotyping silver carp with this chip can perform relatively accurate association analysis on the economic traits of silver carp, helping to locate trait-associated loci and genes.

[0124] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A silver carp 20K liquid-phase chip, characterized in that, The liquid-phase chip includes a probe combination covering 20,909 SNP molecular markers across the whole genome. The physical positions of the 20,909 SNP molecular markers are determined based on sequence alignment of the silver carp genome (GCA_041475455.1), and the locus information of the 20,909 SNP molecular markers is shown in Table 1 of the specification.

2. The silver carp 20K liquid phase chip according to claim 1, characterized in that, Among the molecular markers, 459 are associated with the economic traits of silver carp, and the economic traits are body weight and body height related to growth traits, oxygen consumption rate related to hypoxia tolerance traits, and sex traits; at the same time, the molecular markers also include 460 population-specific loci that can distinguish different varieties of silver carp.

3. A kit for detecting SNP molecular marker combinations of silver carp, characterized in that, It includes the liquid-phase chip as described in Claim 1.

4. Application of a silver carp 20K liquid-phase chip as described in Claim 1 or a kit as described in Claim 2 in the detection of silver carp DNA samples.

5. The application according to claim 4, wherein The application includes the application in genotyping of silver carp samples.

6. The application according to claim 4, characterized in that, The application includes the application in the analysis of the genetic relationship between silver carp and related species.

7. The application according to claim 4, wherein The application also includes the application in the trait association analysis of silver carp breeding materials.

8. A method for preparing a silver carp whole-genome liquid breeding chip, characterized in that, It includes the following steps: (1) Screen high-quality loci from the silver carp resequencing data, and preferably retain loci with high MAF values based on the principle of uniform distribution; (2) Screen SNP loci associated with growth, hypoxia tolerance, and sex traits from the silver carp genome-wide association analysis data, and at the same time obtain specific loci that can distinguish different varieties and the Hubei Jianli population of silver carp from the results of allele frequency difference analysis of the silver carp resequencing data; (3) Remove loci that cannot design probes on both sides of the locus from the above loci, and remove probes that cannot be uniquely aligned on the genome and whose flanking sequences contain repetitive sequences; (4) Design probes for the set of SNP loci that meet the above criteria, construct a probe pool, and obtain a liquid-phase breeding chip for the whole genome of silver carp.

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