Penaeus chinensis 40K liquid phase chip, probe combination and application thereof
By designing a 40K liquid phase chip for Chinese shrimp and employing targeted capture sequencing technology, the problem of insufficient molecular markers was solved, enabling efficient and accurate SNP site detection, improving breeding efficiency, and promoting the protection and utilization of germplasm resources.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
- Filing Date
- 2026-02-13
- Publication Date
- 2026-05-15
AI Technical Summary
In existing technologies, the number of molecular markers for Chinese shrimp is limited, and there is a lack of independently developed high-efficiency liquid phase chips, which restricts the application and development of precision breeding. Furthermore, resequencing technology has high detection costs and poor flexibility, making it difficult to meet the needs of rapid trait aggregation.
A 40K liquid phase chip specifically designed for Chinese shrimp was developed, containing 40,803 SNP sites with comprehensive coverage and good polymorphism. It is used to detect sites related to key traits such as growth, stress resistance, and disease resistance. Targeted capture sequencing technology, combined with probes and reagents, is employed to achieve efficient and accurate SNP site detection.
It improves the efficiency of genetic diversity analysis, trait association analysis, and genome selection breeding of Chinese shrimp populations, promotes the protection and utilization of high-quality germplasm resources, fills the gap in the independent research and development of liquid phase chips, and has important scientific research value and industrial application prospects.
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Abstract
Description
Technical Field
[0001] This invention relates to a 40K liquid phase chip for Chinese shrimp, a probe assembly, and their applications, specifically belonging to the field of aquatic molecular breeding biotechnology. Background Technology
[0002] The Chinese shrimp (Fenneropenaeus chinensis), belonging to the phylum Arthropoda, class Crustacea, order Decapoda, and family Penaeidae, is also known as the Oriental shrimp or Ming shrimp. It is mainly distributed in the Yellow Sea, Bohai Sea, and the northern East China Sea. As one of my country's important marine aquaculture economic species, the Chinese shrimp enjoys a high reputation in domestic and international markets. However, the number of molecular markers for the Chinese shrimp is currently limited, and there is a lack of independently developed high-performance liquid chromatography chips, which restricts the application and development of its precision breeding.
[0003] In recent years, although resequencing technology has been widely used in Chinese shrimp research, its high detection cost and poor flexibility still exist, making it difficult to meet the needs of precision breeding such as rapid trait aggregation. Liquid-phase chips based on target capture sequencing genotype detection technology (GBTS) achieve deep resequencing of target sites by reducing library abundance. They have advantages such as high customization, flexible use, and cost-effectiveness, and are widely used in fields such as genome-wide selection and mapping of genes related to important traits, showing great potential in aquatic animal research. Summary of the Invention
[0004] To overcome the shortcomings of existing technologies, this invention provides a 40K liquid phase chip specifically for Chinese shrimp, containing probes corresponding to 40,803 SNP sites. These sites are evenly distributed on the reference genome ASM1920278v2, providing comprehensive coverage and good polymorphism, and are especially closely related to key traits such as growth, stress resistance, and disease resistance.
[0005] The locations of the SNP molecular marker combinations on the Chinese shrimp reference genome ASM1920278v2 are shown in Table 1. In the table, the first digit in the name indicates the chromosome, the number after the second underscore indicates the physical location, and the colon indicates the reference base. For example, NC_061819.1_10031191: A indicates that the chromosome ID is NC_061819.1, the physical location is 10031191, and the reference base at this site is A.
[0006] Table 1 SNP loci
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[0130] The chip design aims to improve the efficiency of genetic diversity analysis, trait association analysis, and genome selection breeding of Chinese shrimp populations, and to promote the protection and utilization of high-quality germplasm resources.
[0131] The liquid-phase chip includes a primer set and / or probes for detecting the SNP sites, a 40K probe mixture for SNP capture, and a reagent for liquid-phase hybridization capture. The 40K probe mixture contains highly sequence-specific probes for identifying and detecting target SNP sites. The reagent for liquid-phase hybridization capture is used to capture the target SNP sites. The 40K liquid-phase targeted capture probes are designed based on the positive strand of the Chinese shrimp reference genome. The probe length is set to 110 bp, the GC content is controlled between 30% and 70%, and the number of homologous regions does not exceed 5. The conventional regions are regions with low sequence complexity and high uniformity; the highly complex regions are regions containing high GC content, repetitive sequences, or other complex structures.
[0132] This invention also provides a probe array for SNP sites in Chinese shrimp. The probe is used to detect target SNP sites. The probe is designed based on the positive strand of the Chinese shrimp reference genome. The length of the probe is set to 110 bp, the GC content is controlled at 30% to 70%, and the number of homologous regions does not exceed 5.
[0133] The present invention also provides a kit comprising a primer set and / or probes for detecting SNP sites in the liquid phase chip.
[0134] The present invention also provides the application of the Chinese shrimp whole genome 40K liquid phase chip and the kit in the fields of Chinese shrimp variety identification, whole genome breeding or assisted breeding, genetic diversity assessment, genetic map construction, gene localization, molecular marker-assisted selection, genotyping, whole genome association analysis, cluster analysis or kinship identification, and germplasm resource improvement and protection.
[0135] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0136] This invention addresses the current limitations of molecular markers for Chinese shrimp and the lack of independently developed microarrays for the species. It provides a 40K liquid-phase microarray for Chinese shrimp based on targeted capture sequencing. The 40K liquid-phase microarray design of this invention is based on high-depth sequencing data from multiple species populations, providing comprehensive SNP site coverage and rich information. Employing targeted capture sequencing technology, it offers high detection efficiency and accuracy, and is flexible and upgradable. This 40K liquid-phase microarray for Chinese shrimp can simultaneously meet various application needs, including molecular identification, genome selection, and functional gene research. This invention fills the gap in independently developed liquid-phase microarrays for Chinese shrimp and has significant scientific research value and promising prospects for industrial application.
[0137] This invention also provides specific application scenarios for the aforementioned 40K liquid phase chip for Chinese shrimp, including but not limited to: germplasm resource evaluation and variety identification, population genetic structure research and protection, genome selection breeding, genome-wide association analysis and candidate gene mining, etc. Attached Figure Description
[0138] Figure 1 This is a site density map of the 40K SNP chip in the whole genome of Chinese shrimp;
[0139] Figure 2 This is a principal component analysis diagram of 13 populations of Chinese shrimp.
[0140] Figure 3 These are the CV error values corresponding to different K values in population structure analysis;
[0141] Figure 4 It is an ADMIXTURE plot in population structure analysis;
[0142] Figure 5 This is a graph showing the cross-validation results of the accuracy of the genomic selection model;
[0143] Figure 6 It is a Manhattan plot in genome-wide association analysis. Detailed Implementation
[0144] The technical solution of the present invention will be further described in detail below with reference to specific examples. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.
[0145] Example 1: Design and fabrication of a 40K liquid phase chip for Chinese shrimp
[0146] 1. A total of 120 wild Chinese shrimp samples from Changyi and Rizhao, Shandong Province, were selected and sequenced using whole-genome resequencing technology. Each sample generated approximately 10 Gb of data, with a sequencing depth of approximately 7×.
[0147] 2. The 120 resequencing data of Chinese shrimp obtained in step 1 were aligned to the reference genome (ASM1920278v2) and variant detection was performed, initially identifying 29,945,692 SNP loci. Using the selection criteria of MAF ≥ 0.1, deletion rate ≤ 0.1, and heterozygosity ≤ 0.5, 45,580 high-quality SNP loci with even distribution and rich information content were ultimately retained.
[0148] 3. Based on the location and flanking sequence information of the 45,580 SNP sites obtained in step 2, Shijiazhuang Borui Biotechnology Co., Ltd. was commissioned to design primers and synthesize probes using targeted capture sequencing technology. The probe length was set at 110 bp, the GC content was controlled between 30% and 70%, and the number of homologous regions did not exceed 5.
[0149] 4. One hundred muscle samples of Chinese shrimp were randomly selected and tested using the probes designed above. After filtering based on MAF≥0.1, deletion rate≤0.1, and heterozygosity rate≤0.5, 4,777 SNP sites were deleted, leaving 40,803 SNP sites. The detection rate ranged from 98.172% to 99.402%, with an average detection rate of 99.112%. A 40K liquid-phase microarray of Chinese shrimp was prepared, and the distribution of its SNP markers on the genome is shown below. Figure 1 As shown.
[0150] Example 2: DNA extraction and 40K liquid-phase microarray genotyping of Chinese shrimp
[0151] Genomic DNA was extracted from the muscle or other tissues of Chinese shrimp using the phenol-chloroform method or commercial DNA extraction kits. The extracted DNA samples were then subjected to 1%-1.5% agarose gel electrophoresis to check for integrity, and the concentration was determined using a micro-ultraviolet spectrophotometer or similar instrument to adjust the DNA to a working concentration of 10-50 ng / μL. Subsequently, a microarray DNA capture library was constructed using liquid-phase probe hybridization targeting gene capture technology, and sequencing was performed on the BGISEQ2000 sequencing platform. After quality control processing, the raw data underwent SNP detection and genotyping analysis according to standard procedures to support germplasm identification, genomic selection breeding, and population genetics research.
[0152] Example 3: Assessment of population phylogenetic relationships and genetic structure of Chinese shrimp using a 40K liquid phase chip.
[0153] Thirteen Chinese shrimp populations from different geographical regions and environmental conditions were selected, with 10-20 individuals collected from each population. SNP genotyping was performed using the 40K liquid phase chip of this invention. Principal component analysis (PCA) and genetic structure analysis (ADMIXTURE) were then conducted on the 13 populations using these SNP data. The results showed that the 40K liquid phase chip could roughly reflect the phylogenetic relationships of the 13 populations, with the first three principal components explaining 21.08%, 10.23%, and 7.98% of the population variation, respectively. Figure 2 Population genetic structure analysis was performed on populations under different environmental conditions. The results showed that the CV error was minimized when K=3. Figure 3 Therefore, these materials can be divided into 3 subgroups ( Figure 4 ).
[0154] Example 4: Genomic selection breeding of Chinese shrimp using a 40K liquid phase chip
[0155] 1. A reference population with high genetic diversity was established and subjected to salt-alkali stress treatment. Sixty hundred samples with different tolerance levels were collected, and multiple phenotypic data, including body length and weight, were recorded. SNP typing of these samples was performed using the 40K liquid chromatography chip of this invention.
[0156] 2. A genome prediction model was constructed, and multiple methods, including GBLUP, rrBLUP, BayesA, and BayesB, were used to explore the association between genotype and phenotype. The results showed that the Random Forest (RF) method performed best in predicting salt tolerance traits. Figure 5 This method was chosen to estimate the effect size of all labels.
[0157] 3. SNP typing was performed on 600 parent shrimp in the candidate population. The breeding value was estimated based on the constructed prediction model. The top 10% of individuals with the best breeding value were selected for subsequent breeding work.
[0158] Example 5: Genome-wide association analysis using a 40K liquid phase chip for Chinese shrimp
[0159] Vibrio infection experiments were conducted on juvenile Chinese shrimp. Twenty-four surviving shrimp and 24 control shrimp were collected. SNP typing was performed using the 40K liquid-phase chip of this invention. Genome-wide association analysis (GWA) of the SNPs identified by the liquid-phase chip typing was then performed to determine the functional region of the disease resistance trait. The functional region of the disease resistance trait was successfully located on chromosome NC_061829.1. Figure 6 ).
[0160] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.
Claims
1. A 40K liquid phase chip for Chinese shrimp, characterized in that: It contains probes designed for 40,803 SNP sites; the SNP sites are based on the ASM1920278v2 reference genome, and the SNP sites are listed in Table 1 of the specification.
2. The 40K liquid phase chip for Chinese shrimp according to claim 1, characterized in that: The liquid-phase chip includes a primer set and / or probes for detecting the SNP sites, a 40K probe mixture for SNP capture, and a reagent for liquid-phase hybridization capture. The 40K probe mixture contains highly sequence-specific probes for identifying and detecting target SNP sites. The reagent for liquid-phase hybridization capture is used to capture the target SNP sites. The 40K liquid-phase targeted capture probes are designed based on the positive strand of the Chinese shrimp reference genome. The probe length is set to 110 bp, the GC content is controlled between 30% and 70%, and the number of homologous regions does not exceed 5.
3. A probe combination for SNP sites in Chinese shrimp, characterized in that, The probe is used to detect target SNP sites. The probe is designed based on the positive strand of the Chinese shrimp reference genome. The probe length is set to 110 bp, the GC content is controlled between 30% and 70%, and the number of homologous regions does not exceed 5. The SNP sites are shown in Table 1 of the specification.
4. A reagent kit, characterized in that: The kit contains probes or primers for identifying the SNP sites described in Table 1 of the instruction manual.
5. The application of the 40K liquid phase chip for Chinese shrimp as described in claim 1 in the fields of variety identification, whole-genome breeding or assisted breeding, genetic diversity assessment, genetic map construction, gene localization, molecular marker-assisted selection, genotyping, whole-genome association analysis, cluster analysis or kinship identification, and germplasm resource improvement and protection of Chinese shrimp.
6. The probe combination of the SNP sites of Chinese shrimp described in claim 3 is used in the fields of variety identification, whole-genome breeding or assisted breeding, genetic diversity assessment, genetic map construction, gene localization, molecular marker-assisted selection, genotyping, whole-genome association analysis, cluster analysis or kinship identification, and germplasm resource improvement and protection of Chinese shrimp.
7. The application of the kit described in claim 4 in the fields of species identification, whole-genome breeding or assisted breeding, genetic diversity assessment, genetic map construction, gene localization, molecular marker-assisted selection, genotyping, genome-wide association analysis, cluster analysis or kinship identification, and germplasm resource improvement and protection of Chinese shrimp.