Hetian chicken 5K whole genome liquid phase chip based on targeted capture sequencing as well as preparation method and application of Hetian chicken 5K whole genome liquid phase chip
By developing the 5K whole genome liquid chip for Hetian chicken, and utilizing 5781 SNP molecular markers and targeted capture sequencing technology, the problems of high cost and difficulty in implementation of high-density chips in the conservation and breeding of Hetian chicken were solved, achieving low-cost and efficient genetic information detection and breed identification.
Patent Information
- Application Number
- CN202511741405.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-25
- Publication Date
- 2026-01-23
AI Technical Summary
Existing commercial chicken SNP chips are mainly foreign products, which are difficult to detect the unique genetic information of Hetian chickens, such as high-quality meat and disease resistance. In addition, high-density chips are expensive and difficult to implement, and cannot meet the needs of Hetian chicken conservation and breeding.
A liquid-phase chip based on targeted capture sequencing of the Hetian chicken 5K whole genome was developed. Primers were designed and probes were synthesized using 5781 SNP molecular markers. The chip was prepared using targeted capture sequencing technology and is used for Hetian chicken genome selection and preservation, genetic diversity analysis, breed identification and kinship identification.
It achieves low-cost and high-efficiency genome detection for Hetian chickens, providing a basis for genetic diversity analysis, breed identification, and kinship identification. It solves the problems of high cost and difficulty in implementation of high-density chips, and supports the screening of high-quality Hetian chicken breeds.
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Figure CN121380366A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application relates to the technical field of gene chips, in particular to a 5K whole genome liquid chip for river chicken based on targeted capture sequencing and a preparation method and application thereof. BACKGROUND
[0002] As a characteristic local chicken breed in Fujian Province and a national geographical indication product, the core value of river chicken is concentrated in unique meat flavor and strong stress resistance (heat and humidity tolerance, coarse feed tolerance), and these characteristics depend on scarce exclusive genetic resources. At present, river chicken breed source protection is facing two major problems: the risk of genetic purity degradation is increasing and the precision of genetic diversity monitoring is insufficient, and the stability of valuable genetic resources is threatened; in addition, the existing breeding mode of river chicken mainly relies on "phenotypic selection + conventional molecular marker assisted selection", which can maintain basic production performance, but there are obvious technical bottlenecks in the face of high-level requirements such as "multi-trait coordinated improvement", "breeding efficiency improvement" and "hidden trait screening", and it is difficult to realize the precise directional transmission of high-quality traits. At the same time, the river chicken industry is transforming from "scattered small-scale breeding" to "scale, branding and high-end", but it is facing the reality of "lack of standardization, difficulty in releasing brand premium and high cost" - the production end lacks unified quality control standards, and the market end is difficult to prove the purity of "river chicken" through technical means, resulting in high quality but not high price, which restricts the high-quality development of the industry. Therefore, it is necessary to develop a river chicken liquid seed chip, use the chip to preserve the river chicken genome, and obtain high-purity river chicken.
[0003] As the third generation of molecular markers, SNPs have become an indispensable tool in modern genetic research due to their high density, low cost, strong stability, and wide applicability. They can be used in genomics, medicine, agriculture, and forensic science. In the future, with further technological development, the application range and research depth of SNPs will further expand, bringing more breakthroughs in human health, agricultural production, and social security.
[0004] Currently, in the gene chip technology for SNP site detection, traditional solid-phase chips are based on the complementary hybridization of probes and DNA sequences, and are typed by the fluorescence color signals of markers, which have the advantages of high throughput, extremely small amount of use, low unit sample cost in batch detection, but also have some limitations: poor flexibility, complex sample pre-treatment, high pre-equipment and research and development cost. Liquid chip is based on targeted capture resequencing technology, which specifically captures each target site and performs high-depth resequencing, and has the advantages of high detection accuracy, large throughput, flexible application, and high cost performance. At present, it has been widely used in species evolution analysis, germplasm resource evaluation and DNA fingerprint identification, molecular genetic map construction, gene / QTL positioning and gene cloning, molecular marker assisted selection, whole genome selection, etc.
[0005] The existing commercial chicken SNP chips mainly include Illumina 60K, Axiom 600K, Pheno ixChip-I (Illumina 50K) and IASCHICK (Illumina 50K), which have been widely used by breeding enterprises and scientific research units, and have played a key role in chicken genome research and breeding, providing high-density genetic marker data to support whole genome association analysis, population structure research, trait genetic mechanism analysis and genome selection. However, there is still a blank stage for the invention of low-density conservation chip for Hetian chicken, and these existing commercial chicken SNP chips are mainly designed by foreign products, which are difficult to detect the unique genetic information of Hetian chicken, such as high-quality meat quality, strong disease resistance and adaptability, etc. Therefore, how to develop a low-density liquid chip suitable for Hetian chicken conservation is a technical problem that needs to be solved by those skilled in the art. SUMMARY
[0006] The purpose of the present application is to provide a Hetian chicken 5K whole genome liquid chip based on targeted capture sequencing and its preparation method and application, in order to solve the problem of high cost and difficulty in implementation of high-density chip in Hetian chicken conservation breeding process. The Hetian chicken 5K whole genome liquid chip can be used for Hetian chicken genome selection conservation, genetic diversity analysis, variety identification, kinship identification and whole genome association analysis.
[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a Hetian chicken 5K whole genome liquid chip, the detection target point of the Hetian chicken 5K whole genome liquid chip is composed of 5781 SNP molecular markers; the 5781 SNP molecular markers are located on the chicken reference genome GRCg7b in the 7th edition as shown in Table 1; Table 1 .
[0008] The application further provides a preparation method of the river chicken 5K whole genome liquid chip, comprising the following steps: (1) performing whole genome resequencing on the river chicken to obtain original data, and performing comparison between the original data and a chicken reference genome GRCg7b in version 7 to obtain a basic SNP data set; (2) after preliminary filtering of the basic SNP data set, selecting, evaluating and obtaining 5781 SNP molecular markers; (3) according to the position and sequence information of the 5781 SNP molecular markers, designing primers and synthesizing probes by using a targeted capture sequencing technology to obtain the river chicken 5K whole genome liquid chip.
[0009] Preferably, the preliminary filtering method in step (2) is: selecting SNP molecular markers with a minimum sequencing depth of ≥5X, a deletion rate of <50% and a minor allele frequency of >0.05; and removing non-biallelic SNP molecular markers and InDel SNP molecular markers.
[0010] Preferably, the selecting method in step (2) is: selecting SNP molecular markers with a heterozygosity of ≤50%, a deletion rate of ≤10%, a minor allele frequency of ≥0.35 and uniform distribution.
[0011] Preferably, the evaluation principle in step (2) is: the probe length is 110bp, the probe GC content is 30-70%, and the number of homologous regions is ≤5.
[0012] The application further provides application of the river chicken 5K whole genome liquid chip and the river chicken 5K whole genome liquid chip obtained by the preparation method in river chicken conservation.
[0013] The application further provides the application of the river crab 5K whole genome liquid phase chip and the river crab 5K whole genome liquid phase chip prepared by the preparation method in river crab genetic diversity analysis.
[0014] The application further provides the application of the river crab 5K whole genome liquid phase chip and the river crab 5K whole genome liquid phase chip prepared by the preparation method in river crab genetic diversity analysis.
[0015] The application further provides the application of the river crab 5K whole genome liquid phase chip and the river crab 5K whole genome liquid phase chip prepared by the preparation method in river crab genetic diversity analysis.
[0016] The application further provides the application of the river crab 5K whole genome liquid phase chip and the river crab 5K whole genome liquid phase chip prepared by the preparation method in river crab genetic diversity analysis.
[0017] The application has the following beneficial effects: The river crab 5K whole genome liquid phase chip is obtained based on the targeted capture sequencing technology, and the problems of high cost and great implementation difficulty of high-density chips in the process of river crab breeding selection are solved. The river crab 5K whole genome liquid phase chip can be used in river crab genome selection, genetic diversity analysis, variety identification, genetic relationship identification and whole genome correlation molecules, and provides a basis for screening and identifying excellent river crab varieties. BRIEF DESCRIPTION OF DRAWINGS
[0018] Figure 1 Distribution diagram of 5781 SNP molecular markers on each chromosome; Figure 2 River crab 5K whole genome liquid phase chip cluster relationship diagram of 307 river crabs; Figure 3 River crab 5K whole genome liquid phase chip genetic relationship numerical diagram of 307 river crabs; Figure 4 Three-dimensional diagram of the first three PCs of the river crab 5K whole genome liquid phase chip for 307 river crabs. DETAILED DESCRIPTION
[0019] The technical solutions provided by the application are described in detail below in combination with the embodiments, but they should not be understood as limitations on the protection scope of the application.
[0020] Example 1
[0021] Preparation method of river crab 5K whole genome liquid phase chip based on targeted capture sequencing
[0022] Selection of test materials: 50 river field chickens of Longyan Changting in Fujian were selected, half male and half female.
[0023] Extraction of genomic DNA: Blood was collected from river field chickens, and DNA was extracted using a high-throughput DNA extraction kit from Shijiazhuang Boruitai Biotechnology Co., Ltd. The DNA was quality tested using the following two methods: (1) Agarose gel electrophoresis was used to analyze the degradation degree of DNA and whether there was RNA, protein and other contamination; (2) Qubit 2.0 was used to accurately quantify the DNA concentration; after quality detection, it can be used for subsequent cDNA library construction and sequencing.
[0024] Construction of cDNA library: GenoBaits® DNA Library Prep Kit was used to construct the resequencing library of qualified DNA, and the steps were as follows: (1) Take 200 ng of qualified DNA and place it in a 0.2 mL PCR tube. Add 4 μL GenoBaits® End Repair Buffer and 2.6 μL GenoBaits® End Repair Enzyme to the tube, add water to 20 μL, and place it in an ABI 9700 PCR instrument for 37°C incubation for 20 min, 72°C denaturation for 20 min. Complete the processes of DNA fragmentation, end repair and A tailing.
[0025] (2) Add 2 μL GenoBaits® Ultra DNA Ligase, 8 μL GenoBaits® Ultra DNA Ligase Buffer and 4 μL GenoBaits® Adapter, add water to 40 μL, and place it on an ABI 9700 PCR instrument at 22°C for 60 min. Complete the connection of the sequencing adapter. Then add 48 μL of GenoPrep DNA Clean Beads to purify the connection product. After purification, use magnetic beads to screen fragments, and retain the connection product with insert fragments between 300-350 bp.
[0026] (3) Add 10 μL of sequencing adapter with Barcode sequence, 10 μL of GenoBaits® PCR Master Mix to the PCR tube of the previous step, and add water to 20 μL; use ABI 9700 PCR instrument for amplification, get the second round of PCR product, the amplification program is: 98°C pre-denaturation for 2 min; 98°C denaturation for 30 s, 65°C annealing for 30 s, 72°C extension for 40 s, 5 cycles; 72°C extension for 4 min.
[0027] (4) To the second round of PCR product, add 20 μL GenoPrep DNA Clean Beads, place on the magnetic stand until the solution is clear, discard the supernatant and wash the magnetic beads with 100 μL of 80% ethanol, add 35 μL of 10 mM Tris-HCl to obtain the purified DNA library.
[0028] Library quality control and sequencing: The purified DNA library was first quantified by Qubit 2.0, then the effective concentration of the library was accurately quantified by qPCR to ensure the quality of the library. The qualified library was sequenced by Huada DNBSEQ-T7 sequencing platform to obtain raw data, and the sequencing mode was PE150.
[0029] Raw data filtering: Use software fastp (version 0.20.0, parameters: -n10-q20-u840) to filter the raw data, and the data processing steps are as follows: (1) Remove adapter sequences; (2) When the content of N in the sequencing read exceeds 10 bases, the paired reads need to be removed; (3) When the number of low-quality (Q<20) bases in the sequencing read exceeds 40% of the length of the read, the paired reads need to be removed; (4) After quality control of the raw data, use software BWA to align the quality-controlled data with the reference genome sequence GRCg7b (Genome assembly: bGalGal1.mat.broiler.GRCg7b (GCA_016699485.1)) of the 7th edition of chicken. Through alignment, the location of the quality-controlled data on the reference genome can be located.
[0030] SNP variant detection: Use Sentieon standard process for SNP variant detection, a total of 17,763,229 SNPs are detected, which are used as the basic SNP dataset for subsequent analysis.
[0031] Chip background site selection: After the initial filtration of the detected SNP molecular markers (selecting SNP molecular markers with minimum sequencing depth ≥ 5X; deletion rate < 50%; minor allele frequency MAF > 0.05; and removing non-biallelic SNP molecular markers and InDel SNP molecular markers), the following four conditions are selected, and the selection principle is: heterozygosity ≤ 50%, deletion rate ≤ 10%, minor allele frequency MAF ≥ 0.35, and uniformly distributed SNP molecular markers. The obtained SNP molecular markers are evaluated, and the evaluation principle is: probe length 110 bp, probe GC content: 30-70%, number of homologous regions ≤ 5 (homology calculation: a, more than 40 bp identical; b, similarity 85%, length 80 bp; c, similarity 95%, length 70 bp). Finally, 5781 uniformly distributed SNP molecular markers of the river crab chicken genome are obtained, and the specific genomic coordinates are shown in Table 1. The distribution of all sites in the river crab chicken genome of each chromosome is shown in Table 1. Figure 1
[0032] According to the position and sequence information of the 5781 SNP molecular markers, the primers were designed and the probes were synthesized by Shijiazhuang Boruidi Biotechnology Co., Ltd. by using targeted capture sequencing technology, so as to obtain chicken low-density 5K whole genome liquid SNP chip.
[0033] Example 2
[0034] Using the 5K whole genome liquid SNP chip of Example 1 to detect the sample to be tested
[0035] 1. DNA sample extraction: The high-throughput DNA extraction kit from Shijiazhuang Boruidi Biotechnology Co., Ltd. was used to extract DNA from 307 river crab chicken samples from Longyan Changting, Fujian.
[0036] 2. Library preparation using GBTS technology: GBTS can achieve precise capture of genomic target sites and target regions, and can simultaneously detect SNP, InDel, SSR and other genetic variations.
[0037] (1) GenoBaits experimental process: take a quantitative DNA, use restriction endonuclease for fragmentation, and the broken DNA is connected to A tail after end repair. Use ligase to connect the A-added DNA fragments with sequencing adapters, and then use carboxyl-modified magnetic beads to purify the library. The ligation product is added with barcode sequencing primer and high-fidelity PCR reaction system for PCR amplification, and different barcodes are used to distinguish different samples. After purification by carboxyl magnetic beads, the amplification product can be used for probe hybridization experiment. Take 500 ng of the constructed sequencing library, freeze-dry, add probes and hybridization reagents, denature and incubate at 65°C for 2 hours to complete the hybridization reaction. After washing the hybridization product with washing solution, a round of PCR is performed to complete the construction of the hybridization capture library.
[0038] (2) Library construction and sequencing: using the above DNA samples, the corresponding products are used for targeted sequencing library construction, and finally the library preparation of all samples in this project is completed. After the library construction is completed, Qubit2.0 is used for preliminary quantification, and qPCR method is used for accurate quantification of the effective concentration of the library to ensure the quality of the library. After the library detection is qualified, it enters the sequencing stage.
[0039] 3. Sequencing data filtering: The raw data raw reads obtained by sequencing contain adapter-containing, low-quality reads. In order to ensure the quality of information analysis, raw reads must be filtered to obtain clean reads, which are used for subsequent analysis. Use software fastp (version 0.20.0, parameters: -n 10-q 20-u 40) to filter raw reads, and the data processing steps are as follows: Remove adapter sequences; When the content of N in the sequencing read exceeds 10 bases, the paired reads need to be removed; When the number of low-quality (Q≤20) bases in the sequencing read exceeds 40% of the length of the read, the paired reads need to be removed.
[0040] 4. Reference sequence comparison analysis: Use software BWA (mem alignment method) to align the cleaned reads after quality control with chicken reference genome sequence GRCg7b. Through alignment, the location of clean reads on the reference genome can be located.
[0041] 5. Molecular marker filtering: filter the obtained genotype results, and subsequent analysis is based on the filtered markers on autosomes, and the filtering conditions are as follows: (1) Site type filtering, screening molecular markers located on autosomes and not containing InDel.
[0042] (2) Read depth filtering of sites, screening molecular markers with read coverage depth ≥ 5x.
[0043] (3) Completeness filtering: screening molecular markers with genotypes covering at least 90% of individuals. That is, for a single polymorphic marker site, at least 90 out of 100 individuals have a determined genotype. By filtering markers with poor genotype integrity coverage.
[0044] (4) Polymorphic marker filtering: Polymorphic marker ratio (Pn) refers to the proportion of markers showing polymorphism in the target population. Screening markers with minor allele frequency (MAF) ≥ 0.05 as polymorphic markers.
[0045] The final filtered SNP molecular marker results are shown in Table 2.
[0046] Table 2 SNP molecular marker filtering results
[0047] Total: Total number of markers located on autosomes; InDel: Number of markers after filtering InDel; NA: Number of markers after completeness filtering; MAF: Number of markers after minimum allele frequency filtering, that is, number of polymorphic markers; Pn: Polymorphic marker ratio. The proportion of polymorphic markers in total markers.
[0048] 6. Detection reliability analysis: The average detection rate of 307 samples was 99.98%.
[0049] 7. Population genetics index statistics: Higher heterozygosity means more abundant population genetic diversity, and vice versa. When the observed heterozygosity (Ho) is lower than the expected heterozygosity (He), it is inferred that the population has undergone selection or inbreeding; if the observed heterozygosity is higher than the expected heterozygosity, the population may have introduced bloodlines from other varieties. Using Plink (v1.90b6.18) software to calculate Ho and He, it is found that in 307 samples, Ho is 0.3605 and He is 0.37013, and analysis shows that the observed heterozygosity of Heta chicken is lower than the expected heterozygosity, thus meeting the theoretical expectation of "population undergoing selection or inbreeding", which may lead to a slight loss of genetic diversity.
[0050] 8、Clustering analysis: 307 samples of He Tian chicken were clustered using the Neighbor-Joining (NJ) method. From the analysis results, it can be roughly inferred which samples have closer blood relationship. Generally, samples of a family will be grouped together, so from the clustering results, it can be roughly known that there are several families in total and which samples belong to the same family. The NJ tree was constructed using MEGA-X software (model: p-distance; bootstrap: 1000 times), and the results are as follows Figure 2 As can be seen from the figure, the 5K whole genome liquid chip can successfully cluster He Tian chicken into 6 families, and the clustering effect is completely consistent with the resequencing results; this shows that the 5K whole genome liquid chip can well complete the analysis of the clustering relationship of He Tian chicken.
[0051] 9、Relationship identification: The relationship matrix calculated by genetic markers is more real. Since the pedigree information of the breeding population is usually severely missing, the G matrix is obviously more suitable for judging the relationship of the breeding population. The G matrix was calculated by the software GCTA. The numerical distribution of the relationship matrix was statistically plotted, as shown in Figure 3 As can be seen from the figure, most of the relationship is distributed in [-0.1, 0), [0, 0.1), which means that most individuals have no close relationship, mainly distant or no relationship, which shows that the He Tian chicken population is rich in diversity, which is consistent with the resequencing results, proving that the 5K whole genome liquid chip can be used for relationship identification.
[0052] 10、Principal component analysis (PCA): The gcta software was used for PCA analysis to obtain the variance explanation rate of each principal component (PC) and the score matrix of the sample in each PC. The key information extracted from the SNP information is divided into PC1, PC2, PC3... according to the effect from large to small, and the pairwise scatter plot of the first three PCs is mainly displayed. The three-dimensional graph of the first three PCs is shown in Figure 4 (the variance explanation rate corresponding to the PC in the bracket), as can be seen from the figure, the 5K whole genome liquid chip can well detect whether there are multiple varieties of He Tian chicken, which is consistent with the resequencing results, proving that the 5K whole genome liquid chip can be used for He Tian chicken variety identification.
[0053] As can be seen from the above examples, the 5K whole genome liquid chip of He Tian chicken is obtained based on the targeted capture sequencing technology, which solves the problem of high cost and high difficulty in the process of breeding and breeding of He Tian chicken. The 5K whole genome liquid chip of He Tian chicken can be used for He Tian chicken genome selection, genetic diversity analysis, variety identification, relationship identification and whole genome association molecule. It provides a basis for screening and identifying excellent He Tian chicken varieties.
[0054] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.
Claims
1. A river crab 5K whole genome liquid chip, characterized in that, The detection target of the river chicken 5K whole genome liquid chip is composed of 5781 SNP molecular markers; the positions of the 5781 SNP molecular markers on the chicken reference genome GRCg7b in the 7th edition are shown in Table 1; Table 1 。 2. A method for preparing a river crab 5K whole genome liquid chip, characterized in that, Comprise the following steps: (1) Whole genome resequencing is performed on the river chicken to obtain raw data, and the raw data is aligned with the chicken reference genome GRCg7b in the 7th edition to obtain a basic SNP dataset; (2) After primary filtering of the basic SNP dataset, selection, evaluation, 5781 SNP molecular markers are obtained; (3) According to the position and sequence information of the 5781 SNP molecular markers, primers are designed and probes are synthesized by using targeted capture sequencing technology to obtain the river chicken 5K whole genome liquid chip.
3. The preparation method according to claim 2, characterized in that, The method of the primary filtering in step (2) is to select SNP molecular markers with a minimum sequencing depth of ≥5X, a deletion rate of <50%, and a minor allele frequency of >0.05; and to remove non-biallelic SNP molecular markers and InDel SNP molecular markers.
4. The preparation method according to claim 2, characterized in that, The method of the selection in step (2) is to select SNP molecular markers with a heterozygosity rate of ≤50%, a deletion rate of ≤10%, a minor allele frequency of ≥0.35, and uniform distribution.
5. The preparation method according to claim 2, characterized in that, The principle of the evaluation in step (2) is that the probe length is 110 bp, the probe GC content is 30-70%, and the number of homologous regions is ≤5.
6. The river chicken 5K whole genome liquid chip of claim 1 and the river chicken 5K whole genome liquid chip obtained by the preparation method of any one of claims 2-5 are used in river chicken conservation.
7. The river chicken 5K whole genome liquid chip of claim 1 and the river chicken 5K whole genome liquid chip obtained by the preparation method of any one of claims 2-5 are used in river chicken genetic diversity analysis.
8. The river chicken 5K whole genome liquid chip of claim 1 and the river chicken 5K whole genome liquid chip obtained by the preparation method of any one of claims 2-5 are used in river chicken kinship identification.
9. The river chicken 5K whole genome liquid chip of claim 1 and the river chicken 5K whole genome liquid chip obtained by the preparation method of any one of claims 2-5 are used in river chicken breed identification.
10. The river chicken 5K whole genome liquid chip of claim 1 and the river chicken 5K whole genome liquid chip obtained by the preparation method of any one of claims 2-5 are used in river chicken whole genome molecular.