Macrobrachium rosenbergii 80K liquid phase chip and application thereof
By designing the 80K liquid phase chip of M. Rohmann and using the principle of DNA complementarity for genotype analysis, the problem of high cost and low throughput of genotyping of M. Rohmann in the existing technology was solved, and high-throughput, low-cost and flexible genotyping tools were realized, which improved the efficiency and accuracy of breeding work.
Patent Information
- Application Number
- CN202411670545.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-21
- Publication Date
- 2025-04-01
AI Technical Summary
The prior art has high cost, low throughput and inflexibility in the genotyping of M. Rohmannia, which makes it difficult to effectively capture target regional sites, affecting the efficiency and accuracy of breeding work.
A 80K liquid phase chip of M. Rohmannia was designed. Based on the principle of DNA complementarity, a probe covering the target SNP was designed to hybridize with the resequencing library to form double-strands. The genotype of the target site was obtained through elution, amplification, and sequencing, thereby improving detection throughput and flexibility.
High-throughput typing of uniformly distributed SNP sites on the genome of M. Rohbacillus Rohbacillus Rohbacillus is achieved, reducing detection costs, improving detection accuracy and repeatability, and providing a flexible breeding tool.
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Figure CN119736399A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of Macrobrachium rosenbergii molecular breeding, and in particular to a Macrobrachium rosenbergii 80K liquid phase chip and an application thereof. Background Art
[0002] Macrobrachium rosenbergii, as one of the important freshwater economic shrimps, is widely farmed in China, Thailand, Vietnam, Malaysia and other countries. It is mainly farmed in Guangdong, Guangxi Zhuang Autonomous Region, Fujian, Zhejiang, Jiangsu, Hubei, Hunan and other provinces in China. As of 2023, China's aquaculture output has reached nearly 200,000 tons, ranking first in the world in Macrobrachium rosenbergii aquaculture output. However, the limited introduction of original species and long-term intensive production have led to the decline of Macrobrachium rosenbergii germplasm resources, which has seriously troubled the healthy and sustainable development of Macrobrachium rosenbergii aquaculture. Therefore, it is urgent to carry out corresponding breeding work.
[0003] Traditional breeding is mainly based on phenotypic selection, which has a long cycle, high investment and is easily affected by environmental factors. With the continuous development of whole genome SNP typing technology, molecular marker-assisted breeding and whole genome selection breeding are increasingly used in the breeding of aquatic animals. Existing whole genome SNP typing technologies mainly include random sequencing genotyping technologies such as whole genome resequencing and simplified genome sequencing, as well as solid phase chips; their characteristics are as follows:
[0004] 1. Random sequencing genotyping technology obtains genetic variation sites across the entire genome through direct sequencing; however, it is costly, time-consuming, requires large amounts of data analysis, and generates a lot of redundant data, making it unsuitable for large-scale genotyping work.
[0005] 2. Solid-phase chips are based on the complementary base hybridization characteristics. Oligonucleotide probes complementary to the sites to be tested are fixed on a solid carrier. The sites to be tested are captured through hybridization reactions and then single-base extension is performed. Finally, genotype analysis is performed based on the captured fluorescent signals. However, its initial development cost is high and site selection is inflexible. Summary of the invention
[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a Macrobrachium rosenbergii 80K liquid phase chip and its application. Based on the principle of DNA complementarity, the present invention designs probes covering the target SNP at each site to be tested, and these probes are hybridized with the denatured resequencing library to form a double strand. The genotype of the target site is finally obtained through elution, amplification and sequencing, which can greatly increase the detection throughput, reduce the detection cost and improve the detection flexibility. Therefore, the present invention can capture the target region site on Macrobrachium rosenbergii in a targeted manner, and is a Macrobrachium rosenbergii genotyping tool with high throughput, low cost, high detection specificity, strong repeatability and flexible site selection.
[0007] To achieve the above purpose, the technical solution designed by the present invention is as follows:
[0008] The present invention provides a SNP site combination of a Macrobrachium rosenbergii 80K liquid phase chip, wherein the SNP site combination includes 79541 background sites and 582 functional sites, and the physical positions of the background sites and the functional sites on the Macrobrachium rosenbergii genome CNP0006369 are respectively shown in the following two Tables 1 and 2.
[0009] The present invention also provides an application of a chip for detecting the above-mentioned functional sites in analyzing important economic traits of Macrobrachium rosenbergii; the economic traits include one or more of growth-related traits and low-temperature resistance-related traits.
[0010] The present invention also provides a Macrobrachium rosenbergii 80K liquid phase chip, the 80K liquid phase chip comprising a probe combination for detecting the above-mentioned sites; the probe combination comprises two types of capture probes, wherein the first type of capture probes comprises probes for detecting 79541 background sites, and the second type of capture probes comprises probes for detecting 582 functional sites;
[0011] The sites detected by the first type of capture probes are shown in Table 1, and the sites detected by the second type of capture probes are shown in Table 2.
[0012] Furthermore, in the 80K liquid phase chip, the probe design requirements are as follows:
[0013] The design is based on the upstream and downstream sequences of each site, with a length of 100-120 bp, a GC content between 30-70%, and the number of homology regions ≤5.
[0014] The present invention also provides an application of the above-mentioned Macrobrachium rosenbergii 80K liquid phase chip in the genotyping of Macrobrachium rosenbergii.
[0015] The present invention also provides an application of the above-mentioned Macrobrachium rosenbergii 80K liquid phase chip in the genetic structure analysis of Macrobrachium rosenbergii.
[0016] The present invention also provides an application of the above-mentioned Macrobrachium rosenbergii 80K liquid phase chip in Macrobrachium rosenbergii germplasm evaluation, molecular marker-assisted breeding and whole genome selection breeding.
[0017] The present invention also provides an application of the above-mentioned Macrobrachium rosenbergii 80K liquid phase chip in analyzing important economic traits of Macrobrachium rosenbergii; the economic traits include one or more of growth-related traits and low-temperature resistance-related traits.
[0018] Beneficial effects of the present invention:
[0019] 1. The Macrobrachium rosenbergii 80K liquid phase chip of the present invention can realize the simultaneous typing of 80123 SNP sites evenly distributed on the genome of Macrobrachium rosenbergii with a small amount of sample, and is a breeding tool with high throughput, low cost, simple operation, fast analysis speed and less redundant data;
[0020] 2. The Macrobrachium rosenbergii 80K liquid phase chip of the present invention improves the accuracy, repeatability, and specificity of Macrobrachium rosenbergii genotyping, and at the same time improves the flexibility of site selection, providing a powerful tool for the genetic structure analysis, germplasm evaluation, molecular marker-assisted breeding, and other work of Macrobrachium rosenbergii in the future.
[0021] 3. The present invention contains 582 loci significantly associated with growth and cold-resistance traits, and has great utilization value in molecular marker-assisted breeding for growth and cold-resistance traits in the future.
[0022] In summary, the present invention incorporates multiple SNP sites related to important economic traits as functional sites into the core sites of the liquid phase chip, promoting the application of molecular marker-assisted breeding and whole gene selection breeding technology in Macrobrachium rosenbergii. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The distribution diagram of the core sites on the chromosome in Example 1;
[0024] Figure 2 The MAF value distribution diagram of the core site in Example 1;
[0025] Figure 3 This is a diagram of the nucleotide polymorphism results based on 80K liquid phase chip typing data in Example 4;
[0026] Figure 4 This is the phylogenetic tree result diagram based on 80K liquid phase chip typing data in Example 4. DETAILED DESCRIPTION
[0027] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.
[0028] Example 1 Screening of core sites of Macrobrachium rosenbergii
[0029] Carry out whole genome resequencing of Macrobrachium rosenbergii and screen eligible SNP sites based on resequencing data. The specific steps are as follows:
[0030] 1. Whole genome resequencing of Macrobrachium rosenbergii and identification of SNP loci
[0031] Muscle tissues of 115 Macrobrachium rosenbergii individuals were randomly collected, and whole-genome DNA was extracted using a DNA extraction kit. Qualified DNA samples were tested for library construction and resequencing. Fastp (v 0.20.0) software was used to perform quality control and filter the obtained raw sequencing data to obtain high-quality clean data. BWA-MEM (v 0.71563) software was used to map the clean data to the Macrobrachium rosenbergii reference genome (CNP0006369, its website: https: / / db.cngb.org / search / assembly / CNA0142816 / ). GATK software was used to compare the files for variation detection, and finally 154,614,101 SNP sites were obtained for subsequent liquid phase chip SNP site screening.
[0032] 2. Initial screening of core sites
[0033] Based on the resequencing data obtained above, the background sites were initially screened according to the screening conditions of heterozygosity rate less than 50%, deletion rate less than 10%, MAF greater than 0.1, and uniform distribution of sites, and 89,591 background sites that met the conditions were initially screened. According to the results of the whole genome association analysis conducted by the inventor based on the growth and low temperature tolerance phenotype data of Macrobrachium rosenbergii, 608 SNP functional sites related to 20 economic traits such as low temperature death temperature of adult shrimp / juvenile shrimp, cumulative survival time of adult shrimp / juvenile shrimp at low temperature, adult shrimp weight, and adult shrimp body length were selected.
[0034] 3. Final screening of core sites and probe synthesis
[0035] We commissioned Boredy Biotech Co., Ltd. to design and synthesize probes for the above 90,199 SNP sites. The requirements for probe design are as follows:
[0036] Designed based on the upstream and downstream sequences of each site, the length is 110 bp, the GC content is between 30-70%, and the number of homology regions is ≤5;
[0037] Twelve randomly selected Macrobrachium rosenbergii samples were used to test the capture stability of all loci, and 80,123 SNP loci were finally selected. The distribution of the 80,123 core loci on chromosomes is shown in Figure 1 , the MAF value distribution is shown in Figure 2 ; They are the core SNP sites of Macrobrachium rosenbergii 80K liquid phase chip, including 79,541 background sites and 582 functional sites. The physical location information of these background sites and functional sites on the genome are listed in Table 1 and Table 2, respectively. The details of the two tables are as follows:
[0038] Table 1 Physical location of background sites
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[0167] Note: The value on the left of “-” is the chromosome, and the value on the right is the specific position of the background site on the genome of Macrobrachium rosenbergii CNP0006369;
[0168] Table 2 Physical location information of functional sites
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[0172] Note: “:” The value on the left is the chromosome, and the value on the right is the specific location of the functional site on the genome of Macrobrachium rosenbergii CNP0006369.
[0173] Example 2 Based on the SNP site information obtained in Example 1, the Macrobrachium rosenbergii 80K liquid phase chip was developed, as follows:
[0174] The Macrobrachium rosenbergii 80K liquid phase chip includes a probe combination for detecting the above 80,123 core sites; the probe combination includes two types of capture probes, wherein the first type of capture probes includes probes for detecting 79,541 background sites, and the second type of capture probes includes probes for detecting 582 functional sites;
[0175] The sites detected by the first type of capture probes are shown in Table 1, and the sites detected by the second type of capture probes are shown in Table 2.
[0176] The above probe design requirements are as follows:
[0177] The design was based on the upstream and downstream sequences of each site, with a length of 110 bp, a GC content between 30-70%, and the number of homology regions ≤5.
[0178] Example 3 Genotyping using Macrobrachium rosenbergii 80K liquid phase chip
[0179] The liquid phase chip developed in Example 2 was used to genotype 200 randomly selected Macrobrachium rosenbergii. In this example, the reagents used for library construction and hybrid capture were from the GenoBaitsDNA-seq Library Prep Kit of Shijiazhuang Boredi Biotechnology Co., Ltd., including GenoBaits Block I, GenoBaits Block II, GenoBaits2×Hyb Buffer, GenoBaits Hyb Buffer Enhancer, GenoBaits 2×Beads Wash Buffer, GenoBaits 10×Wash Buffer I, GenoBaits 10X Wash Buffer II, GenoBaits 10XWashBuffer III, GenoBaits 10X Stringent Wash Buffer. The specific operations are as follows:
[0180] 1) Genomic DNA extraction: Collect muscle tissue of the sample to be tested, and use a high-throughput DNA extraction kit to extract the sample DNA. The integrity and concentration of the extracted DNA are tested by 1% agarose gel electrophoresis and Nano UV spectrophotometer.
[0181] 2) Library construction: Take quantitative DNA, fragment it by ultrasonication, repair the ends of the broken DNA fragments, add A treatment and connect sequencing adapters, and purify the library by carboxyl modified magnetic beads. Then use sequencing primers with barcodes and high-fidelity PCR reaction system for PCR amplification to obtain the sequencing library for probe hybridization experiments;
[0182] 3) Hybridization capture: Take 500 ng of the sequencing library constructed in step 2), freeze-dry it, add liquid chip probes and hybridization reagents, denature it and incubate it at 65°C for 2 hours for hybridization reaction. After washing the hybridization product, perform another round of PCR to complete the construction of the hybridization capture library.
[0183] 4) Sequencing: Use Qubit2.0 to perform preliminary quantification of the hybridization capture library, and then use qPCR to accurately quantify the effective concentration of the library. After the library is qualified, it is sequenced on the machine.
[0184] 5) Genotype analysis: The original sequencing data obtained was quality controlled. The data quality statistics showed that the average clean reads of 200 samples were about 18,526,030, and the average Q20 and Q30 were 98.62% and 94.75%, respectively. The clean data was compared with the reference genome, and the average comparison rate was 85.54%. Variant detection and genotyping were performed based on the comparison file.
[0185] The final analysis results showed that the average site detection rate of the Macrobrachium rosenbergii developed in Example 2 in 200 experimental individuals was 95.373%, the number of heterozygous sites in all samples was between 4440 and 41626, the number of homozygous mutation sites was between 6528 and 21608, and the number of unmutated sites was between 16352 and 28589. The typing statistics of some individuals are shown in Table 3.
[0186] Table 3 Statistical results of typing of the developed 80K liquid phase chip in some individuals
[0187]
[0188] Example 4 Genetic structure analysis using liquid phase chip
[0189] Using the genotype data obtained from the 80K liquid phase chip in Example 2, the genetic structure of the 200 Macrobrachium rosenbergii individuals in Example 3 was analyzed:
[0190] First, Plink v1.9 software was used for site filtering (parameters: --indep-pairwise 50 10 0.2–maf0.05–hwe 1e-5–max-missing 0.8–min-alleles 2–max-alleles 2). Then, vcftools software was used for nucleotide polymorphism analysis (parameters: --windows-pi 100000–windows-pi-step10000).
[0191] The results showed that the average nucleotide polymorphism of the tested samples was 8.49×10-5( Figure 3 ). The phylogenetic tree was constructed using the neighbor-joining method in phylip software. The results showed that the 200 individuals were divided into five branches ( Figure 4 ).
[0192] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.
Claims
1. A SNP site combination of Macrobrachium rosenbergii 80K liquid phase chip, characterized by: The SNP site combination includes 79541 background sites and 582 functional sites, and the physical positions of the background sites and the functional sites on the Macrobrachium rosenbergii genome CNP0006369 are shown in the following two tables: Table 2 Physical location of background sites Note: The value on the left of "-" is the chromosome, and the value on the right is the specific position of the background site on the genome of Macrobrachium rosenbergii CNP0006369; Physical location information of functional sites Note: The value on the left of ":" is the chromosome, and the value on the right is the specific position of the functional site on the genome of Macrobrachium rosenbergii CNP0006369.
2. Application of a chip for detecting the functional site according to claim 1 in analyzing important economic traits of Macrobrachium rosenbergii; characterized in that: The economic traits include one or more growth-related traits and low-temperature resistance-related traits.
3. A Macrobrachium rosenbergii 80K liquid phase chip, characterized in that: The 80K liquid phase chip includes a probe combination for detecting the sites described in claim 1; the probe combination includes two types of capture probes, wherein the first type of capture probes includes probes for detecting 79,541 background sites, and the second type of capture probes includes probes for detecting 582 functional sites; The sites detected by the first type of capture probes are shown in the physical position information table of background sites in claim 1 , and the sites detected by the second type of capture probes are shown in the physical position information table of functional sites in claim 1 .
4. The Macrobrachium rosenbergii 80K liquid phase chip according to claim 3, characterized in that: In the 80K liquid phase chip, the probe design requirements are as follows: The design is based on the upstream and downstream sequences of each site, with a length of 100-120 bp, a GC content between 30-70%, and the number of homology regions ≤5.
5. Use of the Macrobrachium rosenbergii 80K liquid phase chip according to claim 3 or 4 in genotyping of Macrobrachium rosenbergii.
6. Use of the Macrobrachium rosenbergii 80K liquid phase chip according to claim 3 or 4 in the genetic structure analysis of Macrobrachium rosenbergii.
7. Use of the Macrobrachium rosenbergii 80K liquid phase chip according to claim 3 or 4 in Macrobrachium rosenbergii germplasm evaluation, molecular marker-assisted breeding and whole genome selection breeding.
8. An application of the Macrobrachium rosenbergii 80K liquid phase chip according to claim 3 or 4 in analyzing important economic traits of Macrobrachium rosenbergii; characterized in that: The economic traits include one or more growth-related traits and low-temperature resistance-related traits.