A 20k liquid chip and its application

Abstract

The application discloses a mylopharyngodon piceus 20K liquid phase chip and application thereof, the liquid phase chip comprises a probe combination of 20,909 SNP molecular markers covering the whole genome, physical positions of the 20,909 SNP molecular markers are determined based on mylopharyngodon piceus genome (GCA_041475455.1) sequence alignment, and site information of the 20,909 SNP molecular markers is shown in Table 1 in the specification. The application provides a first whole genome SNP liquid phase breeding chip of mylopharyngodon piceus, effectively reduces the cost of application of mylopharyngodon piceus in genetic diversity analysis, QTL positioning, GWAS analysis and the like in scientific research, solves the problem that there is no applicable product for mylopharyngodon piceus molecular assisted breeding and whole genome selection breeding, and speeds up the process of mylopharyngodon piceus basic research and breeding, and has important significance for improving the utilization efficiency of mylopharyngodon piceus germplasm resources, speeding up the breeding process of excellent traits, and improving the economic value and environmental adaptability of a breeding variety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of gene chip technology, and particularly relates to a Mylopharyngodon piceus 20K liquid chip and application thereof. BACKGROUND

[0002] In the field of aquaculture, as one of the important freshwater cultured fish in China, the yield of Mylopharyngodon piceus occupies a significant position in the fishery economy. However, for a long time, the traditional selection of Mylopharyngodon piceus mainly relies on phenotypic characteristics, which is inefficient and limited in accuracy, and it is difficult to meet the urgent needs of modern fisheries for high-quality and high-yield Mylopharyngodon piceus 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 advantages such as large number, wide distribution, high stability, and easy detection. Through accurate identification and analysis of SNP sites in the whole genome of Mylopharyngodon piceus, its genetic structure and variation rules can be deeply understood, providing key genetic information basis for carrying out molecular marker-assisted breeding of Mylopharyngodon piceus, thereby greatly accelerating the breeding process of Mylopharyngodon piceus excellent varieties.

[0003] Although SNP molecular markers provide a broad prospect for the genetic research of Mylopharyngodon piceus in theory, in actual application, traditional SNP detection methods such as PCR-RFLP, direct sequencing, etc. have drawbacks such as low throughput, high cost, and complicated operation, which makes it difficult to achieve efficient and accurate detection of a large number of samples and numerous SNP sites. As a new high-throughput genotyping platform, liquid chip technology can fix a large number of SNP probes on carriers such as microspheres, and carry out hybridization reaction with DNA samples to be detected in a liquid environment. With its characteristics of high throughput, high accuracy, and strong flexibility, it brings new opportunities to solve the SNP detection problem. The application of liquid chip technology to SNP molecular marker detection of Mylopharyngodon piceus is expected to break through the bottleneck of traditional technology and realize large-scale and accurate analysis of genetic information of Mylopharyngodon piceus.

[0004] At present, the aquaculture industry is facing severe challenges such as increasing resource and environmental constraints and increasingly fierce market competition, and it is urgent to improve the industrial competitiveness through technological innovation. For Mylopharyngodon piceus breeding, cultivating new varieties with fast growth rate, strong disease resistance, excellent meat quality and other comprehensive excellent traits is the core task to realize the sustainable development of the industry. With the help of SNP molecular markers and liquid chip technology, genetic markers closely related to these excellent traits can be accurately screened, and precise molecular design breeding can be carried out. This not only helps to improve the economic and social benefits of Mylopharyngodon piceus breeding, but also has far-reaching strategic significance for ensuring the stable supply and sustainable development of China's freshwater fisheries. SUMMARY

[0005] The main purpose of the present application is to provide a 20K liquid chip for hypophthalmichthys nobilis and its application, aiming to provide a high-throughput, high-accuracy, low-cost, multifunctional, widely applied hypophthalmichthys nobilis molecular marker chip, which can be widely applied to hypophthalmichthys nobilis whole genome association analysis, germplasm resource identification, genetic diversity evaluation, genetic map construction and QTL positioning, molecular marker assisted selection and whole genome selection and other aspects.

[0006] In order to achieve the above purpose, the present application provides a 20K liquid chip for hypophthalmichthys nobilis, which comprises a probe combination covering 20,909 SNP molecular markers of whole genome, the physical position of the 20,909 SNP molecular markers is determined based on the sequence alignment of hypophthalmichthys nobilis genome (GCA_041475455.1), and the site information of the 20,909 SNP molecular markers is shown in Table 1 of the specification.

[0007] Table 1: Site 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 the black carp, the economic traits being the body weight and body height related to growth traits, the oxygen consumption rate related to hypoxia tolerance traits, and the gender traits; and the molecular markers further comprise 460 population-specific loci capable of distinguishing different varieties of the black carp.

[0062] The application further provides a kit for detecting the SNP molecular marker combination of the black carp, comprising the liquid chip as described above.

[0063] The application further provides the use of the 20K liquid chip of the black carp as described above or the kit as described above in the detection of the DNA sample of the black carp.

[0064] Preferably, the use comprises the use in the genotyping of the black carp sample.

[0065] Preferably, the use comprises the use in the analysis of the genetic relationship between the black carp and the close relative species.

[0066] Preferably, the use further comprises the use in the trait association analysis of the breeding material of the black carp.

[0067] The application further provides a preparation method of the whole-genome liquid breeding chip of the black carp, comprising the following steps:

[0068] (1) screening high-quality loci from the resequencing data of the black carp, and reserving high-MAF-value loci based on the principle of uniform distribution;

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

[0070] (3) Remove sites from the above sites where probes cannot be designed on both sides of the site, and remove probes that cannot be uniquely aligned on the genome and contain repetitive sequences in the flanking sequences.

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

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

[0073] (1) This invention provides the first whole-genome SNP liquid phase breeding chip for silver carp, which effectively reduces the cost of silver carp in scientific research applications such as genetic diversity analysis, QTL mapping, and GWAS analysis. It solves the problem of no applicable products for molecular-assisted breeding and whole-genome selection breeding of silver carp, and accelerates the process of basic research and breeding of silver carp. It is of great significance for improving the utilization efficiency of silver carp germplasm resources, accelerating the breeding process of superior traits, and enhancing the economic value and environmental adaptability of farmed varieties.

[0074] (2) Based on the collection of 624 silver carp germplasm resources with rich diversity across the country and 310 breeding population sequencing data, this invention selects 20,909 molecular marker sites that are highly representative, highly polymorphic (MAF mean 0.31), have good universality, high coverage on the genome (average coverage 99.46%), and are evenly distributed. The chip has high detection throughput, large output information, high target site detection rate, and accurate and reliable typing results (reproducibility rate 99.20%). The chip has strong universality. The average detection rate of this 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 phase chip involved in this invention adds 459 functional markers related to traits such as growth, sex, hypoxia tolerance, and oxygen consumption rate of silver carp, which is beneficial for molecular marker-assisted selection, targeted improvement, multi-gene aggregation breeding, and the mining, identification, and functional analysis of important trait genes; at the same time, it adds 460 SNP fingerprint markers for specific varieties to achieve rapid and accurate identification of silver carp varieties, which has great utilization value in the process of silver carp variety improvement and variety selection.

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

[0077] Figure 1 The distribution diagram of the SNP liquid-phase breeding chip loci of silver carp 20K on the chromosome provided by the present invention;

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

[0079] Figure 3 The genotype detection rate of the silver carp 20K SNP liquid phase breeding chip locus provided by the present invention in the sample;

[0080] Figure 4 A genotypic consistency rate diagram of duplicate samples of silver carp 20K SNP liquid-phase breeding chip provided by the present invention;

[0081] Figure 5 Phylogenetic trees were constructed based on the genotyping information of silver carp and different silver carp populations using the silver carp 20K SNP liquid-phase breeding chip provided by this invention.

[0082] Figure 6 To verify the genome-wide association analysis (QQ plot and Manhattan plot) of the hypoxia tolerance trait of silver carp based on the 20K SNP liquid-phase breeding chip provided by this invention. Detailed Implementation

[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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0084] Example 1: Design and fabrication of liquid-phase breeding chip for silver carp whole genome SNP

[0085] 1. Collection of silver carp diversity materials

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

[0087] Table 2. Information on resequencing samples of silver carp

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[0090] 2. Silver carp whole genome resequencing

[0091] The collected silver carp samples underwent DNA extraction and whole-genome resequencing. Specific steps included:

[0092] (1) DNA was extracted from the sample tissue using magnetic beads. (2) The DNA sample was fragmented using dsDNAFragmentase, the enzyme ends were repaired, and an A base was added to the 3' end. (3) The sequencing adapter and fragmented DNA were linked using ligase, and the ligation product was purified using magnetic beads. (4) The ligation product was amplified using PCR, and the PCR product was screened for fragments using magnetic beads. (5) The linear library was denatured into single strands and then circularized. After digesting the uncirculated linear DNA molecules, a single-stranded circular library was obtained. (6) The single-stranded circular DNA molecules were replicated through rolling circle replication to form a DNA nanosphere (DNB) containing more than 300 copies. (7) The DNB was loaded into a sequencing chip using the MGIDL-T7 loading device, and sequencing was performed using a combined probe anchoring polymerization technique. 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 workflow is as follows:

[0094] (1) Sentieon was used to align reads to the corresponding silver carp reference genome (GCA_041475455.1), orient them, and mark duplicate reads. (2) Sentieon was used to detect variant sites for each sample to obtain variant information for each sample. (3) Sentieon was used to perform joint-calling and joint analysis of gVCF for all samples to obtain the variant results for each individual in the population. To ensure the accuracy of SNPs, preliminary hard filtering was performed 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 loci

[0097] The MAF value, detection rate, heterozygosity, and sequencing depth of the loci were calculated and statistically analyzed from the VCF file containing SNP variant information for all samples. Polymorphic SNP loci with MAF ≥ 0.1, SNP detection rate ≥ 90%, heterozygosity ≤ 40%, and sequencing depth ≥ 10× were selected as initial candidate loci. All candidate loci were used for probe design, with probes designed for each target locus within a 100bp range upstream and downstream. Probe lengths were approximately 100bp, and GC content ranged from 20% to 80%. Based on the probe design results, probes that could not be uniquely aligned to the genome or contained repetitive sequences in their flanking sequences were removed. Based on the principle of uniform distribution, loci with high MAF values ​​were preferentially retained, ultimately yielding 19,990 highly polymorphic whole-genome background loci.

[0098] (2) Functional sites

[0099] a. Seven loci related to silver carp growth traits and 131 loci related to hypoxia tolerance traits were collected from previous studies.

[0100] b. Sex phenotypic data from 105 resequencing samples were collected and organized. Locus population indices in the VCF files containing all variation information from the 105 silver carp samples were calculated and statistically analyzed. Loci with a deletion rate greater than 0.1%, MAF values ​​less than 0.05, and those located in contigs were filtered out. Genome-wide association analysis was performed using a mixed linear model (MLM), based on a threshold p = 1e -5 210 loci that were significantly associated with sex were identified.

[0101] c. Growth phenotypic data from 97 resequencing samples of *Cyprinus longfengensis* and 100 resequencing samples of *Cyprinus spp.* were collected and processed. Genome-wide association analysis was performed using the R package rMVP, which included three models: General Linear Model (GLM), Mixed Linear Model (MLM), and FarmCPU model. The kinship matrix and principal components were added as covariates to the model for correction, reducing the influence of kinship and population structure. The threshold line p = 1e was used. -5 By combining the results of the three models, 111 sites that were significantly associated with growth traits were finally located.

[0102] (3) Population-specific sites

[0103] Genotypes of 108 individuals from four populations—common silver carp, Changfeng silver carp, the new Changfeng silver carp strain 2, and the Hubei Jianli population—were extracted from the above resequencing data. Genotypes with SNP detection rates greater than or equal to 90%, MAF greater than or equal to 0.05, and dimorphism were first screened. Then, differential loci among varieties were screened based on differences in allele distribution, ultimately yielding 460 variety-specific differentiation loci.

[0104] After integrating and deduplicating all the loci from (1) to (3) above, a set of 20K SNP liquid-phase breeding microarray loci for silver carp was formed, with a final number of 20,909 loci (Table 1). These loci have an average coverage of 99.70% on the silver carp chromosome, an average spacing of 38Kb, and the 20K loci are evenly distributed on each chromosome. The density distribution diagram is shown below. Figure 1 As shown in the figure. The silver carp 20K SNP microarray locus exhibits high polymorphism, with a mean minor allele frequency (MAF value) of 0.31. The MAF value distribution is shown in the figure. Figure 2 As shown.

[0105] The 20,909 SNP loci screened were used to develop a liquid-phase breeding chip for silver carp 20K SNPs using Huazhi's independently developed liquid-phase probe precise localization sequencing and genotyping technology (cGPS). cGPS is based on an optimized thermodynamic stability algorithm model, which designs specific probes for target region sequences. Then, the synthesized specific probes are used to capture and enrich multiple different target sequences located at different genomic positions through liquid-phase hybridization. The captured and enriched target genomic sequences are then used to construct sequencing libraries and perform high-throughput sequencing to obtain the genotypes of all SNP / InDel loci within the target region.

[0106] Example 2: Application of Silver Carp 20K SNP Liquid Phase Breeding Chip in Silver Carp Genotyping

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

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

[0109] DNA was extracted from the samples using a magnetic bead method, and the DNA samples underwent quality testing. Quality testing included determining DNA concentration using a Qubit quantitative PCR instrument and assessing DNA integrity using 1% agarose gel electrophoresis. Samples that passed quality control were used for library preparation.

[0110] 2. cGPS Experiment and Analysis Procedure

[0111] (1) Take 200 ng of qualified genomic DNA, digest the DNA sample with fragmentation enzyme, repair the enzyme ends and add an A base to the 3' end, and detect the fragment size by agarose gel electrophoresis.

[0112] (2) Using T4 ligase, the adapter fragments were ligated to both ends of the DNA, and the ligation products were purified using fragment sorting magnetic beads. The concentration of the purified products was detected using a Qubit real-time fluorescence instrument, and the fragment size was detected by agarose gel electrophoresis;

[0113] (3) The purified ligation products were subjected to PCR amplification, and the amplified products were screened for fragments using magnetic beads. The concentration of the screened products was detected by a Qubit real-time fluorescence instrument, and the fragment size was detected by agarose gel electrophoresis.

[0114] (4) Place the qualified library, blocking reagent, RNase inhibitor, and 20K liquid phase breeding chip probe on a PCR instrument for hybridization reaction and incubate at 55℃ for 16-24 hours.

[0115] (5) The hybridization product was captured by streptavidin, the captured library was amplified and enriched, and PE150 sequencing was performed using the BGI sequencing DNBSEQ-T7 platform.

[0116] (6) The raw data after high-throughput sequencing underwent quality control filtering and other processing. FASTP software was used to remove adapter fragments and low-quality reads, resulting in high-quality Clean Reads. BWA software was used to align the obtained Clean Reads with the reference genome and sort them by position to obtain the sorted BAM file. GATK software was used to analyze the sequenced results for variant sites, obtaining the genotyping results for the target loci.

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

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

[0119] Sequencing and data analysis revealed that the locus detection rates of 95 silver carp samples ranged from 98.50% to 99.57%, with an average detection rate of 99.03%; the locus detection rates of 3 bighead carp samples ranged from 91.78% to 91.98%, with an average detection rate of 91.87%. (See details below.) Figure 3 The genotypic consistency rate of the technically replicated samples ranged from 98.90% to 99.40%, with an average consistency rate of 99.21%. See details... Figure 4 By comparing the cGPS liquid-phase chip results with the resequencing results of 13 resequencing samples, the genotypic concordance rate ranged from 95.13% to 96.96%, with an average concordance rate of 95.89%. These data indicate that the silver carp 20K SNP liquid-phase breeding chip provides high target locus detection rate, good stability, and accurate and reliable genotyping results for the tested materials.

[0120] Example 4: Application of the Silver Carp 20K SNP liquid-phase breeding chip in the analysis of phylogenetic relationships between silver carp and closely related species.

[0121] Genotyping of 82 silver carp samples and 3 bighead carp samples from different sources was performed using the 20K SNP liquid-phase breeding chip prepared in Example 1 (specific operation method is described in Example 2). Genotyping results of 85 samples were extracted, and the genetic distance matrix was calculated using the IBS method in Plink software, followed by cluster analysis to construct a phylogenetic tree. The results showed that the tested materials were mainly divided into 6 subgroups, and the classification effect was consistent with the actual classification. Figure 5 As shown, the 20K SNP liquid-phase breeding chip for silver carp can effectively distinguish silver carp from different sources and can be used to determine the phylogenetic relationships, evolutionary relationships, and structural composition between different materials.

[0122] Example 5: Application of the Silver Carp 20K SNP liquid-phase breeding chip in trait correlation analysis of silver carp breeding materials

[0123] Six-month-old Changfeng silver carp from the same breeding population were subjected to hypoxia stress. The 107 fish that first exhibited rollover under hypoxia stress were collected as the hypoxia-sensitive group, while the 116 fish that rolledover last were collected as the hypoxia-tolerant group. Genotyping of these 223 Changfeng silver carp was performed using a constructed silver carp 20K liquid-phase breeding chip (see Example 2). The genotyping results underwent quality control, and low-quality loci were removed, resulting in 19257 high-quality SNP loci. Subsequently, a genome-wide association analysis (GWAS) was performed on the genotype information and hypoxia tolerance phenotype (a binary trait of sensitivity and tolerance) of these 223 individuals at these loci using a mixed linear model in Gemma software. The Bonferroni test was used to screen for significant loci, ultimately identifying 7 SNP loci significantly associated with the hypoxia tolerance trait in Changfeng silver carp. The QQ plot and Manhattan plot of the GWAS for hypoxia tolerance are shown below. Figure 6 As shown in the figure. Annotation of these loci revealed that three SNP loci located on chromosomes 5, 8, and 22 were all located at gene loci. A total of 32 candidate genes were identified from these seven loci. Among them, five genes, including prdx1, ent1, and nrf2, showed differential expression in the heart or gill tissues of silver carp under hypoxic stress. These genes are closely related to stress response, DNA damage repair, immune regulation, and hypoxia-inducible factor regulation. Therefore, genotyping of silver carp using this microarray can provide a more accurate association analysis of economic traits, helping to locate trait-related loci and genes.

[0124] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within 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 array of 20,909 SNP molecular markers covering the entire genome. The physical locations of the 20,909 SNP molecular markers are determined based on sequence alignment of the silver carp genome GCA_041475455.

1. The site information of the 20,909 SNP molecular markers is shown in Table 1 of the specification.

2. The silver carp 20K liquid phase chip as described in claim 1, characterized in that, Among the molecular markers, 459 are associated with economic traits of silver carp, including growth traits such as body weight and height, hypoxia tolerance traits such as oxygen consumption rate, and sex traits; in addition, the molecular markers also include 460 population-specific loci that can distinguish different silver carp species.

3. A kit for detecting silver carp SNP molecular marker combinations, characterized in that, Including the liquid phase chip as described in claim 1.

4. The application of the silver carp 20K liquid phase chip as described in claim 1 or the kit as described in claim 3 in the genotyping of silver carp samples.

5. The application of the silver carp 20K liquid phase chip as described in claim 1 or the kit as described in claim 3 in the analysis of the phylogenetic relationship between silver carp and closely related species.

6. The application of the silver carp 20K liquid phase chip as described in claim 1 or the kit as described in claim 3 in trait association analysis of silver carp breeding materials.

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