Macrobrachium rosenbergii 50K SNP liquid phase chip based on targeted capture sequencing and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a macrobrachium rosenbergii 50K SNP (Single Nucleotide Polymorphism) liquid-phase chip based on targeted capture sequencing and application of the liquid-phase chip. The liquid chip provided by the invention provides an important technical support for macrobrachium rosenbergii germplasm resource genotype accurate identification, population genetic diversity evaluation, paternity test and pedigree reconstruction, and excellent functional gene mining. The development of the chip also provides a breeding tool with high precision, low cost and rapid analysis for genome breeding, gene editing and the like, realizes the breakthrough of the domestic macrobrachium rosenbergii breeding chip in China, lays a good foundation for precise breeding of freshwater shrimps, and brings a deep influence on the development of related breeding industries. According to the method, not only is the breeding technology of the macrobrachium rosenbergii improved, but also positive influences are generated on the aspects of improving the breeding efficiency, reducing the cost, enhancing the disease resistance and the like, and powerful scientific and technological support is provided for sustainable development of the macrobrachium rosenbergii industry.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a 50K SNP liquid chip for Macrobrachium rosenbergii based on targeted capture sequencing and its application. Background Art

[0002] Macrobrachium rosenbergii is an important economic shrimp species and is widely cultured globally. China is one of the main countries for the culture and consumption of Macrobrachium rosenbergii, and the Zhejiang region is the largest production base of Macrobrachium rosenbergii fry in the country. However, due to long-term culture and the phenomenon of germplasm degradation (such as increased diseases, slowed growth rate, decreased stress resistance, etc.), it has seriously affected the high-quality development of the Macrobrachium rosenbergii industry.

[0003] Single nucleotide polymorphism (SNP) has the characteristics of large quantity, wide distribution, easy to quickly screen on a large scale, and convenient for genotyping. It is the third-generation genetic marker after the first-generation restriction fragment length polymorphism marker and the second-generation microsatellite, i.e., simple tandem repeat marker, and is considered the best marker choice at present, with important biological significance. At present, SNP markers have become important tools for biological population identification, genetic structure analysis, functional gene mapping, and genomic selection. With the development of high-throughput SNP genotyping technology, methods based on whole-genome or reduced-representation genome sequencing have become the mainstream of SNP genotyping technology. For species with reference genome sequences, researchers generally design SNP chips using known sequences, and hybridize randomly fragmented genomic DNA fragments with oligonucleotide probes on the chip to obtain the SNP genotyping of corresponding sites.

[0004] Genotyping by targeted capture sequencing (GBTS) is a technology that realizes deep re-sequencing only of target sites by reducing library abundance, and it can significantly reduce the cost of genotyping. There are already commercial Macrobrachium rosenbergii chips, but there are still disadvantages such as low genotyping utilization rate and high cost. Summary of the Invention

[0005] The purpose of the present invention is to provide a 50K SNP liquid chip for Macrobrachium rosenbergii based on targeted capture sequencing and its application to solve the problems existing in the above-mentioned prior art. The present invention provides specific SNP probe design principles and a 50K liquid chip for the whole genome of Macrobrachium rosenbergii, which can solve the problems of high cost, inflexibility, and inability to be used on a large scale in Macrobrachium rosenbergii farms in the prior art.

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

[0007] The present invention provides a SNP locus combination for the variety identification of Macrobrachium rosenbergii, including the SNP loci shown in Table 4; the NCBI accession number of the reference genome of the SNP loci is GCA_040412425.1. The whole genome sequence number of Macrobrachium rosenbergii is GCA_040412425.1, and this genomic data has been deposited in NCBI GenBank, and the RefSeq number of this genome is GCF_040412425.1.

[0008] The SNP loci of the present invention are obtained by screening SNP loci by aligning the whole genome sequencing results of Macrobrachium rosenbergii to the reference genome of Macrobrachium rosenbergii.

[0009] The present invention provides a molecular probe combination for detecting the above SNP locus combination, and the molecular probe combination detects the above SNP locus combination in a test sample.

[0010] The molecular probe combination of the present invention is a DNA double-stranded probe combination, which is a nucleotide sequence designed and synthesized according to the screened SNP loci.

[0011] The present invention provides the application of the above SNP locus combination or the above molecular probe combination in any one of the following:

[0012] (1) Application in the germplasm resource identification of Macrobrachium rosenbergii;

[0013] (2) Application in the variety identification of Macrobrachium rosenbergii;

[0014] (3) Application in the whole genome selection breeding of Macrobrachium rosenbergii;

[0015] (4) Application in the paternity testing of Macrobrachium rosenbergii;

[0016] (5) Application in the genetic diversity assessment of Macrobrachium rosenbergii;

[0017] (6) Application in the economic trait analysis of Macrobrachium rosenbergii.

[0018] The present invention provides the application of the above SNP locus combination or the above molecular probe combination in the preparation of a 50K SNP liquid chip for the variety identification of Macrobrachium rosenbergii.

[0019] The present invention provides a 50K SNP liquid chip for the variety identification of Macrobrachium rosenbergii, and the 50K SNP liquid chip includes the above molecular probe combination.

[0020] The present invention provides the application of the above 50K SNP liquid chip in any one of the following:

[0021] (1) Application in the germplasm resource identification of Macrobrachium rosenbergii;

[0022] (2) Applications in the variety identification of Macrobrachium rosenbergii;

[0023] (3) Applications in the genome-wide selection breeding of Macrobrachium rosenbergii;

[0024] (4) Applications in the paternity testing of Macrobrachium rosenbergii;

[0025] (5) Applications in the assessment of genetic diversity of Macrobrachium rosenbergii;

[0026] (6) Applications in the analysis of economic traits of Macrobrachium rosenbergii.

[0027] The present invention provides a method for variety identification of Macrobrachium rosenbergii, comprising the following steps:

[0028] (1) Obtaining genomic DNA of an individual Macrobrachium rosenbergii to be tested;

[0029] (2) Based on the genomic DNA, constructing a sequencing library;

[0030] (3) Performing a probe hybridization reaction between the sequencing library and the above-mentioned 50K SNP liquid chip;

[0031] (4) Extracting the genotyping information after sequencing the sequences captured by the liquid chip to form a genotyping file;

[0032] (5) Obtaining the variety identification result according to the genotyping file.

[0033] The present invention discloses the following technical effects:

[0034] In order to improve the accuracy and efficiency of breeding, the applicant took the lead in uniting several domestic enterprises to develop a 50K cGPS high-precision liquid breeding chip for Macrobrachium rosenbergii with independent Chinese intellectual property rights, namely the 50K SNP liquid chip provided by the present invention. The present invention has made important progress in the breeding technology of Macrobrachium rosenbergii. This 50K SNP liquid chip provides important technical support for the accurate identification of the genotypes of Macrobrachium rosenbergii germplasm resources, the evaluation of the genetic diversity of populations, paternity testing and pedigree reconstruction, and the excavation of excellent functional genes. The development of this chip also provides a high-precision, low-cost and rapidly analyzable breeding tool for genomic breeding, gene editing, etc., achieving a zero breakthrough in domestic breeding chips for Macrobrachium rosenbergii in China, laying a good foundation for the precise breeding of freshwater shrimps, and will have a profound impact on the development of related aquaculture industries. The breeding research of Macrobrachium rosenbergii also includes the collection of germplasm resources and the cultivation of the seed industry to improve the supply capacity of high-quality seedlings. By introducing wild germplasm resources from abroad, carrying out work such as isolation, quarantine, identification, preservation and testing, and conducting research on the correlation between traits and major genes, identifying the key genes of major traits, constructing a genomic selection breeding technology system, screening and cultivating new varieties of Macrobrachium rosenbergii with single or compound traits such as high yield, fast growth, salt tolerance or high temperature resistance, and realizing industrial application. It can be seen that the present invention not only improves the breeding technology of Macrobrachium rosenbergii, but also has a positive impact on improving aquaculture efficiency, reducing costs, enhancing disease resistance, etc., providing strong scientific and technological support for the sustainable development of the Macrobrachium rosenbergii industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] 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 to be used 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 also be obtained based on these drawings.

[0036] Figure 1 is the locus chromosome distribution map; wherein, the abscissa is the chromosome; the ordinate is the number of loci;

[0037] Figure 2 is the marker distribution density map on the chromosome;

[0038] Figure 3 is the MAF distribution map; wherein, the abscissa is the MAF value; the ordinate is the quantity;

[0039] Figure 4It is a statistical chart of SNP mutation types; among them, the abscissa is the SNP type; the ordinate is the quantity; A / C is the number of SNPs with A / C transversions; A / G is the number of SNPs with A / G transversions; A / T is the number of SNPs with A / T transitions; C / G is the number of SNPs with C / G transversions; C / T is the number of SNPs with C / T transversions; G / T is the number of SNPs with G / T transversions;

[0040] Figure 5 It is a distribution map of functional sites; among them, the abscissa Type is the gene structure type, and the ordinate Number is the number of target sites under the relevant type; downstream is downstream; excnic is exon; intergenic is within the gene interval; intronic is intron; splicing is splicing; upstream is upstream; upstream; downstream is upstream, downstream;

[0041] Figure 6 It is the PCA obtained according to the original genotypes of the population;

[0042] Figure 7 It is the PCA obtained according to the SNP sites on the chip;

[0043] Figure 8 It is the phylogenetic tree made according to the original data of the population;

[0044] Figure 9 It is the phylogenetic tree made according to the SNP data on the chip. Detailed implementation manners

[0045] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0046] It should be understood that the terms described in the present invention are only for describing special implementation manners and are not used 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. Each 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.

[0047] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0048] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0049] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0050] Example 1 Liquid Chip Design

[0051] 1. Liquid Chip Information

[0052] Jointly by Zhejiang Institute of Freshwater Fisheries and Beijing Compson Agricultural Technology Co., Ltd., a high-density gene chip applicable to the breeding of Macrobrachium rosenbergii was developed using the target capture technology with independent intellectual property rights in our country. This chip covers functional loci related to various economic traits such as growth and reproduction, and can be widely applied to molecular breeding work such as whole-genome selection breeding, paternity testing, and genetic relationship sorting of Macrobrachium rosenbergii, as well as scientific research such as genetic diversity analysis, QTL, and genome-wide association study (GWAS), providing strong technical support and guiding basis for the variety breeding work of Macrobrachium rosenbergii.

[0053] 2. Sample Information

[0054] The sample information is shown in Table 1.

[0055] Table 1 Sample Information

[0056]

[0057] 3. Chip Locus Screening and Determination

[0058] For the selection of loci on the gene chip, existing VIP functional loci (accumulated from previous research), GWAS-mined functional loci, and variety-specific loci obtained from selection signal analysis are preferentially retained, followed by background loci with uniform genome coverage. Generally, 2 - 4 times of locus selection is recommended.

[0059] 4. Locus Screening Principle​

[0060] (1) Cover relevant loci, such as QTL, GWAS, domestication-related genes, differences between interspecific and intraspecific subgroups, excellent traits, terminators, alternative splicing, non-synonymous mutations and other loci;

[0061] (2) High polymorphism (default MAF ≥ 0.05);

[0062] (3) Uniform genome-wide coverage;

[0063] (4) Multiple-fold locus selection: Generally, 2 - 4 times the number of target loci are selected as the candidate locus set;

[0064] (5) Determine the final chip customization loci according to the scoring of the Compson probe design system.

[0065] 5. Locus selection strategy

[0066] Rank the variant loci by priority to determine the loci to be customized on the chip. The locus selection priorities are as follows:

[0067] (1) Priority 1: VIP loci of important traits, QTL loci, GWAS loci, important genes, common genes, etc.;

[0068] (2) Priority 2: Terminators on annotated genes (select all, but refer to the MAF value), alternative splicing (select all, but refer to the MAF value), non-synonymous mutation sites (select all, but refer to the MAF value), etc.;

[0069] (3) Priority 3: Genome-wide coverage loci.

[0070] 6. Locus quality control

[0071] Perform genotyping verification and data quality control on the variant loci to obtain accurate and high-quality variant loci. The quality control content is shown in Table 3, including: read coverage sequencing depth, deletion rate, kinship detection, minor allele frequency (MAF), Hardy-Weinberg equilibrium, outlier samples, etc. After deleting low-quality samples and loci, 143 samples and 33,348,418 loci remain, as shown in Table 2, for subsequent chip locus selection.

[0072] Table 2 Sample information table

[0073] Variety / Strain Original Sample Size (tails) Remaining Sample Size (tails) Macrobrachium rosenbergii 368 143 Total 368 143

[0074] Table 3 Quality control parameters and thresholds

[0075]

[0076]

[0077] 7. Locus scoring

[0078] Submit the selected candidate site set to the Kangpusen probe design system for scoring, and design probes based on the evaluation results of the upstream and downstream sequences of the target sites. Mainly evaluate the specificity, complexity, GC content, etc. of the upstream and downstream sequences of the target sites. Prioritize placing the target sites in the middle position of the probes, and the designed probes are 120bp in length. According to the scoring results, a total of 50,000 high-quality chip custom sites are selected. After confirmation, the final target site set of the chip is obtained, that is, the 1-fold site set.

[0079] The specific information of the sites is shown in Table 4.

[0080] Table 4 Information of 50,000 SNP Sites

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[0567] Note: The NCBI accession number of the reference genome is GCA_040412425.1.

[0568] 8. Locus Evaluation

[0569] Evaluate the set of target loci for the custom chip at 1x. The main contents include SNP locus profiles, SNP chromosomal distributions, MAF distributions, SNP type distributions, functional locus distributions, SNP density distributions, etc. of the final chip product. The locus evaluation results show that the loci cover the entire genome evenly, cover important trait loci, and can be applied to gene screening, gene mapping, marker-assisted breeding, etc.

[0570] 8.1. Locus Set Ranking

[0571] The locus set ranking is shown in Table 5.

[0572] Table 5 Locus Set Ranking Table

[0573] Tilling_order Number of Loci Proportion Tilling_order1 2229 4.458％ Tilling_order2 47503 95.006％ Tilling_order3 268 0.536％ sum 50000 100％

[0574] Note: Tilling_order1: VIP, functionally annotated loci, GWAS loci, important genes, common genes, etc.; Tilling_order2: loci covering the entire genome; Tilling_order3: loci near indels.

[0575] 8.2. Locus Chromosomal Distribution

[0576] Map the selected loci using the R package. From Figure 1 It can be seen from the above table the priority distribution of the variant loci on each chromosome. Therefore, the distribution of the loci with each priority on the chromosome of this chip is relatively even and the coverage is good.

[0577] 8.3. Locus Density Distribution

[0578] The following figure is the distribution density map of the chip locus set on each chromosome. As Figure 2As shown, the locus distribution of each chromosome is relatively uniform, indicating that the loci contained in this chip can cover each chromosome well.

[0579] 8.4, MAF Distribution

[0580] MAF is the minor allele frequency, which usually refers to the occurrence frequency of uncommon alleles in a given population. For example, for the three genotypes TT, TC, and CC, if the frequency of C in the population is 0.36 and the frequency of T is 0.64, then allele C is the minor allele, and the minor allele frequency MAF = 0.36. Therefore, as Figure 3 shown, the proportion of loci with minor allele frequency > 0.1 is 98.558%.

[0581] 8.5, Mutation Spectrum Analysis

[0582] Base transition refers to the substitution between purines or between pyrimidines; base transversion refers to the substitution between purines and pyrimidines.

[0583] Due to the base structure, the probability of transition usually is higher than that of transversion. After detecting and filtering to obtain high-confidence SNPs, statistical analysis is performed on the SNP mutation spectrum. The results are as Figure 4 shown. Point mutations include 6 types: A / T, A / C, A / G, G / C, G / T, G / A. For example, A / C represents the mutation type from A to C on one strand. By classifying and counting the mutation results of all samples, the percentage of each mutation type in each sample can be seen.

[0584] 8.6, Statistical Distribution of Marked Functional Loci

[0585] The results are as Figure 5 shown. As Figure 5 can be seen, the liquid-phase chip covers key genes and the regulatory regions upstream and downstream of genes, such as exons, introns, alternative splicing regions, etc.

[0586] 8.7, PCA Population Structure

[0587] PCA analysis shows the clustering of individuals within a population in the form of a graph. The genetic structure of the population can be roughly understood through the results of PCA analysis. The results are as Figure 6 and Figure 7 shown. On the PCA graph, usually, the closer the individuals are in terms of the straight-line distance in space, the closer their genetic relationship; the farther the individuals are in terms of the straight-line distance in space, the farther their genetic relationship. For chips containing variety identification loci, good identification effects can be obtained. Figure 6 This is the PCA obtained based on the original genotypes of the population. Figure 7The PCA obtained based on the SNP sites of the chip shows consistent sample aggregation in both cases, indicating that the chip can better represent the original population status.

[0588] Figure 8 and Figure 9 is the visualization result of the phylogenetic tree, where Figure 8 is the phylogenetic tree constructed based on the original population data, Figure 9 is the phylogenetic tree constructed based on the chip SNP data. The sample aggregation is consistent in both cases, indicating that the chip can better represent the original population status.

[0589] Example 2 Application of the liquid chip designed in Example 1

[0590] I. Sample collection and preparation

[0591] 1. DNA extraction:

[0592] For tissue and blood samples, the magnetic bead method is used for DNA extraction. The CWE9600 Magbead Blood DNA Kit from ComWin Biotech is used. Based on the principle that silica-based magnetic beads specifically adsorb DNA, enzymatic hydrolysis is used to release the blood genome, and DNA molecules are bound in a specific and efficient lysis buffer system. By changing the environmental ionic strength, genomic DNA separation and purification are achieved. Add an appropriate amount of tissue sample to a 2.0 mL lysis tube, add Proteinase K and Buffer WL, place it in a vortex mixer and shake for 30 s, then lysate in a 56 °C water bath for 40 min, shaking once every 20 min. After lysis is complete, place it in a centrifuge for instantaneous centrifugation, add Lysate, Buffer KL, and Magbeads PN solution. Insert the Spintips Pack (magnetic rod sleeve) into a 96 DW deep well plate and run the DNA extraction program to extract gDNA.

[0593] 2. Detection:

[0594] 2.1. 0.8% DNA agarose gel electrophoresis is used to analyze the degree of DNA degradation and whether there is RNA contamination;

[0595] 2.2. Nanodrop is used to detect the purity of DNA (OD 260 / 280 ratio);

[0596] 2.3. Qubit is used to accurately quantify the DNA concentration;

[0597] The concentration of genomic DNA is accurately measured by Qubit, and the purity of the DNA sample is detected by Nanodrop (OD 260 / OD 280=1.7-2.1), and agarose gel electrophoresis was used to detect the integrity of the genomic DNA and determine whether the DNA was degraded.

[0598] 2. SNP typing of genomic DNA

[0599] Qualified gDNA (concentration ≥ 50ng / μL) was extracted, and whole genome amplification was first performed on all samples, incubated at 37°C for 20-24h, and then the gDNA was fragmented, precipitated, and resuspended in hybridization buffer. The resuspended DNA fragments were added to the chip, hybridized, and incubated at 48°C for 16-24h. After hybridization, non-specifically bound DNA was removed by washing, and the remaining specifically bound sites were subjected to single base extension. After staining, the data was scanned and read using the Illumina iScan Reader.

[0600] The raw data obtained by scanning with the iScan system was imported into the Illumina official data analysis process IlluminaBead ArrayFiles for data analysis, including standardization, clustering and genotyping of the raw data. And by exporting the Plink format file, .ped and .map files were generated for subsequent analysis. The raw data of the liquid phase chip designed in Example 1 is a file that stores the raw signal value of the SNP probe of the illumina SNP chip. This type of data must be opened using the Illumina official genuine software (Genome Studio).

[0601] Remark:

[0602] 1) Laboratory environment requirements:

[0603] Ambient temperature: 20-25℃; Ambient humidity: 40%~60%; Please wear personal protective equipment and pay attention to laboratory safety.

[0604] 2) Instruments used in the experiment:

[0605] The components of the kit should avoid repeated freezing and thawing. Unless otherwise specified, the preparation and addition of the reaction system during the experiment should be carried out on an ice / low-temperature metal bath to ensure enzyme activity. The laboratory ambient temperature should be stabilized at 20-25°C.

[0606] Example 3

[0607] The liquid phase chip developed in Example 1 was used to identify 285 test samples (core population of Macrobrachium rosenbergii) for testing the detection effect of the liquid phase chip, and the detection method was the same as in Example 2. The results showed that the detection rate of the sample site was 97.42%, and the typing consistency of repeated samples was 99.25%.

[0608] Example 4

[0609] Using the liquid chip developed in Example 1 to identify 48 test samples (the core population of Macrobrachium rosenbergii), among which 1 sample was repeated 3 times, and the detection method was the same as that in Example 2. The results showed that the average detection rate of 50K loci reached 95.4%, and the genotyping consistency of the repeated samples reached 99.25%.

[0610] 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 locus combination for species identification of Macrobrachium rosenbergii, characterized in that: Including the SNP sites shown in Table 4 of the specification; the NCBI accession number of the reference genome of the SNP site is GCA_040412425.

1.

2. A molecular probe combination for detecting the SNP site combination according to claim 1, characterized in that: The molecular probe combination detects the SNP site combination of claim 1 in the sample to be tested.

3. Use of the SNP site combination according to claim 1 or the molecular probe combination according to claim 2 in any of the following: (1) Application in identification of germplasm resources of Macrobrachium rosenbergii; (2) Application in species identification of Macrobrachium rosenbergii; (3) Application in whole genome selection breeding of Macrobrachium rosenbergii; (4) Application in parentage identification of Macrobrachium rosenbergii; (5) Application in the assessment of genetic diversity of Macrobrachium rosenbergii; (6) Application in the analysis of economic traits of Macrobrachium rosenbergii.

4. Use of the SNP locus combination according to claim 1 or the molecular probe combination according to claim 2 in the preparation of a 50K SNP liquid phase chip for species identification of Macrobrachium rosenbergii.

5. A 50K SNP liquid phase chip for species identification of Macrobrachium rosenbergii, characterized in that: The 50K SNP liquid phase chip comprises the molecular probe combination according to claim 2.

6. Use of the 50K SNP liquid phase chip according to claim 5 in any of the following: (1) Application in identification of germplasm resources of Macrobrachium rosenbergii; (2) Application in species identification of Macrobrachium rosenbergii; (3) Application in whole genome selection breeding of Macrobrachium rosenbergii; (4) Application in parentage identification of Macrobrachium rosenbergii; (5) Application in the assessment of genetic diversity of Macrobrachium rosenbergii; (6) Application in the analysis of economic traits of Macrobrachium rosenbergii.

7. A method for identifying species of Macrobrachium rosenbergii, characterized in that: The following steps are involved: (1) Obtaining genomic DNA of the individual Macrobrachium rosenbergii to be tested; (2) constructing a sequencing library based on the genomic DNA; (3) performing probe hybridization reaction between the sequencing library and the 50K SNP liquid phase chip according to claim 5; (4) extracting genotyping information after sequencing of the sequence captured by the liquid phase chip to form a genotyping file; (5) Obtaining variety identification results based on the genotyping file.

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