Macrobrachium rosenbergii whole genome liquid phase chip and application thereof

Abstract

The invention discloses a macrobrachium rosenbergii whole genome liquid phase chip and application thereof, and belongs to the technical field of biology. The invention provides 5010 SNP loci and molecular markers thereof, and a liquid phase chip constructed based on the molecular markers, and the SNP loci achieve complete coverage of macrobrachium rosenbergii genome polymorphic loci. The SNP liquid-phase chip prepared by combining the SNP site with sequences on two sides can be used for carrying out rapid genetic typing on a sample and carrying out genetic identification and variety breeding on macrobrachium rosenbergii, can also be used for screening candidate genes to analyze the correlation between phenotypes and functional alleles, and is an effective method for realizing molecular marker-assisted breeding.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a whole-genome liquid chip for Macrobrachium rosenbergii and its application. Background Art

[0002] Macrobrachium rosenbergii grows fast, has a relatively large body size, good meat quality and is easy to breed, and thus occupies an important position in the freshwater aquaculture industry. With the development of the Macrobrachium rosenbergii aquaculture industry, phenomena such as slow growth of shrimp bodies, miniaturization of individuals and an increase in diseases have emerged, which have an adverse impact on the healthy development of the Macrobrachium rosenbergii aquaculture industry.

[0003] Single nucleotide polymorphism (SNP) has become an indispensable tool in the genotyping process due to its large quantity, wide distribution and suitability for rapid large-scale screening. As the third-generation genetic marker, SNP inherits the development achievements of the first-generation restriction fragment length polymorphism (RFLP) and the second-generation microsatellite marker (SSR), and is recognized as the best genetic marker at present, having important biological value. The liquid chip technology based on SNP has high detection sensitivity and flexibility, and its application in molecular breeding of aquatic animals has been gradually recognized. However, specific chips for Macrobrachium rosenbergii are still scarce in the current market, which restricts its genetic improvement and the development of excellent germplasm resources.

[0004] Therefore, it is necessary to provide an efficient whole-genome liquid chip applicable to Macrobrachium rosenbergii. Summary of the Invention

[0005] The purpose of the present invention is to provide a whole-genome liquid chip for Macrobrachium rosenbergii and its application to solve the problems existing in the above-mentioned prior art. The present invention provides 5010 SNP loci and their molecular markers, as well as a liquid chip constructed based on these molecular markers. These SNP loci achieve complete coverage of the polymorphic loci of the Macrobrachium rosenbergii genome. The SNP liquid chip prepared by using the SNP loci in combination with the sequences on both sides can quickly genotype samples, conduct kinship identification and variety breeding for Macrobrachium rosenbergii, and can also be used to screen candidate genes to analyze the correlation between phenotypes and functional alleles, which is an effective method for realizing molecular marker-assisted breeding.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] The present invention provides a combination of SNP molecular markers for detecting the whole genome of Macrobrachium rosenbergii, including 5010 SNP molecular markers. The SNP locus information corresponding to the SNP molecular markers is shown in Table 1 - Table 12, and the information of the SNP loci is represented in the form of chromosome number: physical position:

[0008] Table 1 Location Information of SNP Loci

[0009]

[0010] Table 2 Location Information of SNP Loci

[0011]

[0012] Table 3 Location Information of SNP Loci

[0013]

[0014] Table 4 Location Information of SNP Loci

[0015]

[0016] Table 5 Location Information of SNP Loci

[0017]

[0018] Table 6 Location Information of SNP Loci

[0019]

[0020] Table 7 Location Information of SNP Loci

[0021]

[0022] Table 8 Location Information of SNP Loci

[0023]

[0024] Table 9 Location Information of SNP Loci

[0025]

[0026] Table 10 Location Information of SNP Loci

[0027]

[0028] Table 11 Location Information of SNP Loci

[0029]

[0030] Table 12 Location Information of SNP Loci

[0031]

[0032] The reference genome version number of the said SNP loci is ASM4041242v1.

[0033] The present invention also provides a probe combination for detecting the above SNP molecular marker combination, and the probe combination can hybridize with the sequence fragment where the above SNP molecular marker combination is located;

[0034] The probes in the probe combination have a length of 110 bp, a GC content of 30%-80%, and the number of homologous regions ≤5.

[0035] The present invention also provides the application of the above SNP molecular marker combination or the above probe combination in preparing a liquid chip for detecting the genome of Macrobrachium rosenbergii.

[0036] The present invention also provides a liquid chip for detecting the whole genome of Macrobrachium rosenbergii, and the liquid chip contains the above probe combination.

[0037] The present invention also provides the application of the above SNP molecular marker combination, the above probe combination or the above liquid chip in the kinship identification or variety identification of Macrobrachium rosenbergii.

[0038] The present invention also provides the application of the above SNP molecular marker combination, the above probe combination or the above liquid chip in the molecular assisted breeding of Macrobrachium rosenbergii.

[0039] The present invention also provides the application of the above SNP molecular marker combination, the above probe combination or the above liquid chip in the genetic diversity analysis or genetic map construction of Macrobrachium rosenbergii.

[0040] The present invention also provides the application of the above SNP molecular marker combination, the above probe combination or the above liquid chip in the analysis of phenotypic trait association of Macrobrachium rosenbergii.

[0041] The present invention also provides a method for genotyping the whole genome of Macrobrachium rosenbergii, which is characterized by including the following steps:

[0042] (1) Extract the genomic DNA of the to-be-detected Macrobrachium rosenbergii;

[0043] (2) Based on the genomic DNA, construct a sequencing library;

[0044] (3) Perform a probe hybridization reaction on the sequencing library and the above liquid chip;

[0045] (4) After sequencing the fragments captured by the liquid chip, obtain the genomic typing data.

[0046] The present invention discloses the following technical effects:

[0047] In the present invention, three major new varieties / lines of Macrobrachium rosenbergii on the market were selected for re-sequencing analysis to obtain a basic locus pool of selected points. Subsequently, 5010 SNP locus combinations of Macrobrachium rosenbergii were screened, achieving complete coverage of the polymorphic loci of new varieties / lines of Macrobrachium rosenbergii. The SNP liquid chip can be prepared by using these SNP loci combined with the flanking sequences, thereby quickly genotyping samples; it can also be used to screen SNP marker loci related to the growth traits of Macrobrachium rosenbergii, laying a foundation for finding candidate genes for the growth traits of Macrobrachium rosenbergii, analyzing the correlation between phenotypes and functional alleles using candidate genes, studying the regulation mechanism of growth traits, and subsequent molecular marker-assisted breeding research; it can also be used for kinship identification and variety breeding of Macrobrachium rosenbergii. The SNP locus combination and liquid chip of Macrobrachium rosenbergii provided by the present invention have important applications in large-scale genotyping, providing an efficient gene typing technology for genetic diversity analysis, strain identification, marker-assisted breeding, etc. of Macrobrachium rosenbergii. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0049] Figure 1 It is a distribution density map of SNP loci involved in the SNP liquid chip of Macrobrachium rosenbergii;

[0050] Figure 2 It is a MAF statistical chart of SNP loci involved in the SNP liquid chip of Macrobrachium rosenbergii;

[0051] Figure 3 It is a Q-Q plot for GWAS analysis of the body weight of Macrobrachium rosenbergii; among them, A is the Q-Q plot obtained by analysis with a simple linear model (GLM); B is the Q-Q plot obtained by analysis with a mixed linear model (MLM); C is the Q-Q plot obtained by analysis with a fixed random model cyclic probability unification (FarmCPU);

[0052] Figure 4 It is a Manhattan plot for GWAS analysis of the body weight of Macrobrachium rosenbergii; among them, A is the Manhattan plot obtained by analysis with a simple linear model (GLM); B is the Manhattan plot obtained by analysis with a mixed linear model (MLM); C is the Manhattan plot obtained by analysis with a fixed random model cyclic probability unification (FarmCPU). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0053] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0055] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention's specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention's specification are obvious to those skilled in the art. The present invention's specification and examples are merely exemplary.

[0056] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0057] Example 1 Construction and Preliminary Verification of a SNP Liquid Chip for Macrobrachium rosenbergii

[0058] 1. Detection Population

[0059] Two common new varieties of Macrobrachium rosenbergii in the market, "Nantaihu No. 3" and "Shufeng No. 1", and a new strain "Zhelan No. 1" were collected. The total number of samples was 135 shrimps, including 50 breeding shrimps of "Nantaihu No. 3", 35 breeding shrimps of "Shufeng No. 1", and 50 breeding shrimps of "Zhelan No. 1".

[0060] 2. Experimental Methods

[0061] 2.1 DNA Extraction and Quality Inspection

[0062] Genomic DNA was extracted from the muscle tissue of Macrobrachium rosenbergii using the GenoBaits kit. After extraction, the DNA concentration was detected using a Qubit concentration meter, and agarose gel electrophoresis was performed to detect the DNA integrity to ensure that the DNA concentration was above 50 ng / μL and there was no obvious degradation. The qualified samples were placed in a 4°C refrigerator for storage and standby.

[0063] 2.2 Sequencing Library Construction

[0064] A sequencing library was constructed using a DNA sequencing library construction kit (Huazhi Biotechnology Co., Ltd., model HZL0150). The specific steps were as follows: The extracted genomic DNA was fragmented by ultrasonic waves to construct a high-throughput sequencing library. An A-tail was added to the ends of the fragmented DNA, and sequencing adapters were ligated. Subsequently, PCR amplification was performed, and the amplified product was purified to obtain the sequencing library. The library concentration was quantitatively detected using Qubit, and it was required that the total library amount reached more than 500 ng.

[0065] 2.3 Design and synthesis of SNP probes

[0066] Based on the whole-genome sequencing data of Macrobrachium rosenbergii (version number ASM4041242v1), SNP sites with polymorphisms in different populations were screened out. Further, corresponding biotin-labeled SNP probes were designed and synthesized according to the SNP sites. The design principle was to design and synthesize probes for the target region sequence based on an optimized thermodynamic stability algorithm model. The probe length was 110 bp, the GC content was controlled between 30% and 80%, and the number of homologous regions was ≤5. In this way, the hybridization stability of the sequence where each SNP was located could be effectively improved, and the efficiency of fragment capture could be increased. At the same time, the specificity of the flanking sequences of the sites on the genome was fully considered to ensure the site-specific capture rate and reduce the sequencing cost.

[0067] A total of 5010 excellent sites (including trait-associated sites) were screened and extracted, and the distribution density was as Figure 1 shown, and the MAF distribution was as Figure 2 shown. The physical positions of each SNP site on the reference genome of Macrobrachium rosenbergii are shown in Table 1 - Table 12. Among them, SNP ID represents the chromosome and position information of the SNP site. For example, "1:135833" indicates the position at 135833 on chromosome 1; Allele is the gene polymorphism of this site.

[0068] According to the position and flanking sequence information of the SNP sites in Table 1 - Table 12, primers were designed and probes were synthesized by Huazhi Biotechnology Co., Ltd. using targeted capture sequencing technology to prepare the Macrobrachium rosenbergii SNP liquid chip.

[0069] 2.4 Targeted capture and sequencing

[0070] The constructed sequencing library was mixed with biotin-labeled SNP probes, and the DNA fragments containing the target SNPs were captured through a magnetic bead capture system using the high affinity between biotin and streptavidin. The captured fragments were subjected to PCR amplification and purification, and finally high-throughput sequencing was performed on a sequencing platform to obtain the genotyping data of the target region.

[0071] 3. Data processing

[0072] 3.1 Data quality control and alignment

[0073] The sequencing data was quality-controlled by FastQC to remove low-quality reads and adapter-contaminated sequences. The retained high-quality data was aligned to the Macrobrachium rosenbergii reference genome using the BWA software to obtain the sequencing information of SNP sites.

[0074] 3.2 SNP Genotyping and Statistics

[0075] The GATK software was used to perform SNP genotyping analysis on the sequencing data, screening out high-quality SNP sites, calculating the allele frequencies of each SNP site in different populations, and evaluating the polymorphism of SNP sites.

[0076] 4. Results and Analysis

[0077] The experimental results showed that the Macrobrachium rosenbergii 5010 liquid chip designed by the present invention could efficiently capture the genomic SNP sites of Macrobrachium rosenbergii. In three new varieties / lines of Macrobrachium rosenbergii populations, the minimum MAF of all sites was greater than 0.05, and the average MAF was greater than 0.25, effectively avoiding the problems of uneven marker density and poor polymorphism that may be caused by the reduced-representation genome sequencing genotyping technology.

[0078] Example 2 Production Test and Verification of Macrobrachium rosenbergii SNP Liquid Chip

[0079] The purpose of this example was to test the effect (such as site coverage rate, accuracy, etc.) of the liquid chip constructed in Example 1 on genotyping randomly collected samples. The specific process was as follows:

[0080] 1. Test Samples

[0081] Samples were collected from a local wet market (commercially sold shrimp). After identification, their genetic relationships were close to those of Nantaihu No. 3 and Shufeng No. 1.

[0082] 2. Experimental Methods

[0083] 2.1 DNA Extraction and Quality Inspection

[0084] Genomic DNA was extracted from the muscle tissue of Macrobrachium rosenbergii using the GenoBaits kit. After extraction, the DNA concentration was detected using a Qubit fluorometer, and agarose gel electrophoresis was performed to detect the DNA integrity to ensure that the DNA concentration was above 50 ng / μL and there was no obvious degradation. The qualified samples were placed in a 4°C refrigerator for storage and standby.

[0085] 2.2 DNA Library Construction

[0086] The extracted genomic DNA was fragmented by ultrasonic waves to construct a high-throughput sequencing library. An A-tail was added to the ends of the fragmented DNA, sequencing adapters were ligated, and then PCR amplification was performed. The amplified products were purified to obtain the sequencing library. The concentration of the library was quantitatively detected using Qubit, and the total amount of the library was required to reach more than 500 ng.

[0087] 2.3 Targeted capture and sequencing

[0088] The constructed sequencing library was mixed with the biotin-labeled SNP probes in the liquid-phase chip of Example 1. Through the magnetic bead capture system, using the high affinity between biotin and streptavidin, the DNA fragments containing the target SNPs were captured. The captured fragments were subjected to PCR amplification and purification, and finally high-throughput sequencing was performed on the sequencing platform to obtain the genotyping data of the target region.

[0089] 3. Data processing

[0090] 3.1 Data quality control and alignment

[0091] The sequencing data was quality-controlled by FastQC to remove low-quality reads and adapter-contaminated sequences. The retained high-quality data was aligned to the Macrobrachium rosenbergii reference genome using the BWA software to obtain the sequencing information of SNP sites.

[0092] 3.2 SNP genotyping and statistics

[0093] The GATK software was used to perform SNP genotyping analysis on the sequencing data, and the SNP site data and genotyping results of all samples were combined.

[0094] 4. Results and analysis

[0095] Production tests were carried out on 140 individual Macrobrachium rosenbergii samples. 26 technical replicates were set up in the experiment (the repeated sample numbers were suffixed with -re), and a total of 166 sample data were generated. The results are shown in Table 13 - Table 15. Through sequencing and data analysis, the site detection rates of the 166 samples were between 98.06% and 99.88%, and the average site detection rate was 98.92%; the site heterozygosity rates were between 24.73% and 30.84%, and the average site heterozygosity rate was 28.70%; the detection results of the repeated samples were highly consistent.

[0096] Table 13 Statistical table of site detection results of Macrobrachium rosenbergii samples

[0097]

[0098] Table 14 Statistical table of site detection results of Macrobrachium rosenbergii samples

[0099]

[0100] Statistical Table of Detection Results of Sample Loci of Macrobrachium rosenbergii

[0101]

[0102] 5. Comparison between Resequencing Technology and Detection Results of SNP Liquid Chip of Macrobrachium rosenbergii

[0103] The above 140 Macrobrachium rosenbergii samples were genotyped using the whole-genome resequencing technology, and the genotype consistency of the common loci was compared with the genotyping data obtained from the liquid chip constructed in Example 1. The results showed that the consistency of the two results could reach 99.5%. This indicates that the liquid chip constructed in Example 1 has high accuracy.

[0104] Example 3 Application of SNP Liquid Chip of Macrobrachium rosenbergii

[0105] To verify the practicability of the SNP liquid chip of Macrobrachium rosenbergii constructed in Example 1, a small-scale genome-wide association study (GWAS) was designed and completed in this example. This study took the growth traits of "Zhe Blue No. 1" as the research object, and through genotyping 1,550 individuals, the genomic regions significantly related to the target traits were identified. The specific process is as follows:

[0106] 1. Sample Selection

[0107] Randomly select 1,550 individuals with phenotypic data from the breeding population in 2024.

[0108] 2. Genotyping

[0109] Using the liquid chip of Example 1, all samples were genotyped through a high-throughput sequencing platform. For the specific process, refer to "2. Experimental Method" and "3. Data Processing" in Example 2. The genotyping success rate reached an average of 98.5% (the number of genotyping failure loci was less than 100).

[0110] 3. Phenotypic Data

[0111] Measure the weight data of 1,550 individuals and perform quality control on the data.

[0112] 4. Data Analysis

[0113] Perform quality control on the genotype data (SNP missing rate < 5%, sample missing rate < 10%, allele frequency > 0.05). Apply the rMVP package and use simple linear model (GLM), mixed linear model (MLM), and fixed random model cyclic probability unification (FarmCPU) analysis to control population structure and kinship, and identify SNP loci significantly related to the target traits. Perform regional annotation on the significant loci to explore their possible functional genes.

[0114] 5. Results

[0115] The results are as Figure 3 and Figure 4 shown. The results show that among 5,010 SNPs, multiple SNPs were found to be significantly associated with the target trait (p < 1×10 5 ), and these loci were distributed on multiple chromosomes across the whole genome, indicating that this method can effectively detect phenotype-related variations.

[0116] The above results indicate that the SNP liquid chip of Macrobrachium rosenbergii constructed in Example 1 can quickly and accurately provide genotyping data, support the genetic analysis of the target population, and provide important gene resources for subsequent breeding. This example proves that conducting GWAS based on the 5,010 SNP loci provided by the present invention and the SNP liquid chip of Macrobrachium rosenbergii constructed in Example 1 not only significantly improves the research efficiency, but also successfully identifies genomic regions significantly associated with the target trait through high-coverage and high-accuracy genotyping information, providing a scientific basis for optimizing breeding strategies.

[0117] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A SNP molecular marker combination for detecting the whole genome of Macrobrachium rosenbergii, characterized in that, It includes 5010 SNP molecular markers; the SNP site information corresponding to the SNP molecular markers is as follows, and the information of the SNP site is expressed in the form of chromosome number: physical position: The reference genome version number of the SNP site is ASM4041242v1.

2. A probe combination for detecting the SNP molecular marker combination according to claim 1, characterized in that, The probe combination is capable of hybridizing with the sequence fragment where the SNP molecular marker combination according to claim 1 is located; The probes in the probe combination are 110 bp in length, have a GC content of 30%-80%, and have a number of homology regions ≤5.

3. Use of the SNP molecular marker combination according to claim 1 or the probe combination according to claim 2 in preparing a liquid phase chip for detecting the genome of Macrobrachium rosenbergii.

4. A liquid-phase chip for detecting the whole genome of Macrobrachium rosenbergii, characterized in that, The liquid phase chip comprises the probe combination according to claim 2.

5. Use of the SNP molecular marker combination according to claim 1, the probe combination according to claim 2 or the liquid phase chip according to claim 4 in kinship identification or species identification of Macrobrachium rosenbergii.

6. Use of the SNP molecular marker combination according to claim 1, the probe combination according to claim 2 or the liquid phase chip according to claim 4 in molecular assisted breeding of Macrobrachium rosenbergii.

7. Use of the SNP molecular marker combination according to claim 1, the probe combination according to claim 2 or the liquid phase chip according to claim 4 in genetic diversity analysis or genetic map construction of Macrobrachium rosenbergii.

8. Use of the SNP molecular marker combination according to claim 1, the probe combination according to claim 2, or the liquid phase chip according to claim 4 in association analysis of phenotypic traits of Macrobrachium rosenbergii.

9. A method for genotyping the whole genome of Macrobrachium rosenbergii, characterized in that, The steps include: (1) extracting genomic DNA of the Macrobrachium rosenbergii to be tested; (2) constructing a sequencing library based on the genomic DNA; (3) performing a probe hybridization reaction between the sequencing library and the liquid phase chip according to claim 4; (4) After sequencing the fragments captured by the liquid phase chip, genome typing data is obtained.

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