Application of SNP (Single Nucleotide Polymorphism) site combination, molecular probe group for detecting SNP site combination or liquid phase chip containing molecular probe group in duck breeding

Abstract

The invention relates to the technical field of genetic breeding, in particular to an application of an SNP (Single Nucleotide Polymorphism) site combination, a molecular probe group for detecting the SNP site combination or a liquid phase chip containing the molecular probe group in duck breeding. The invention discloses an SNP locus combination, a molecular probe group for detecting the SNP locus combination and a liquid phase chip containing the molecular probe group by screening common SNP loci and specific SNP loci of three duck species of Jinding duck, Shaoxing duck and Fujian mountain sheldrake, and the SNP locus combination, the molecular probe group and the liquid phase chip can be used for duck species screening, breeding or identification. The invention further provides a method for identifying the Jinding duck, the Fujian mountain sheldrake or the Shaoxing duck population, and an important detection method and means are provided for establishing a rapid and automatically-operated experimental duck genetic quality detection technical system.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic breeding, and in particular to an application of a SNP site combination, a molecular probe group for detecting the SNP site combination, or a liquid phase chip containing the molecular probe group in duck breeding. Background Art

[0002] With the increasing demand for high-quality experimental animals, specific pathogen-free (SPF) animals have become an important experimental animal resource due to their clear genetic background and lack of specific pathogen infection. When ducks are used as experimental animals, the genetic characteristics and genetic stability of duck breeds are crucial to their quality evaluation, and are an important prerequisite for ensuring the reliability and repeatability of animal test results in disease research. However, the key technical system for standardized quality evaluation of experimental ducks is not yet mature. Duck breeding mainly relies on morphological differentiation. Although this method is effective to a certain extent, as the duck population reproduces from generation to generation, the appearance characteristics of different breeds become more and more similar, making them difficult to distinguish. In some studies, there is even the phenomenon of using the wrong breed, which has poor accuracy, long selection cycle and low efficiency.

[0003] Molecular methods provide new tools and methods for duck breeding, which helps to gain a deeper understanding of the genetic characteristics of duck populations. Genome sequencing technology can fully reveal genetic variation and study the regulatory mechanism of gene expression, but its high application cost and complex data processing limit its application in large-scale breeding. In contrast, SNP detection, as an important means of genetics and medical research, has many advantages. Therefore, it is of great significance to explore a combination of SNP sites for duck breeding. Summary of the invention

[0004] In view of the technical problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a SNP site combination, a molecular probe set for detecting the SNP site combination, or a liquid phase chip containing the molecular probe set for use in duck breeding. The SNP site combination, molecular probe set or liquid phase chip provided by the present invention can be used to screen, identify and breed Jinding ducks, Fujian mountain ducks and Shaoxing ducks.

[0005] The first aspect of the present invention provides an application of a SNP site combination, a molecular probe group for detecting the SNP site combination, or a liquid phase chip containing a molecular probe group in duck breeding; the SNP site combination comprises 6000 SNP sites; the physical position of the SNP site is determined based on the duck reference genome GCF_015476345.1_ZJU1.0_genomic comparison; the SNP site information is shown in Table 1.

[0006] Furthermore, the duck breeding is duck screening, duck selection or duck identification.

[0007] Further, the duck breed is Jinding Duck or Fujian Mountain Shelduck or Shaoxing Duck.

[0008] The second aspect of the present invention provides a method for identifying a Jinding Duck population, comprising the following steps:

[0009] Extract the DNA of the sample to be tested; use the above-mentioned liquid-phase chip to test the sample to be tested, obtain the detection status of all SNP sites and count the number of successfully detected SNP sites among all SNP sites; when the proportion of the number of successfully detected SNP sites in the number of all SNP sites in Table 1 is between 66.83% and 73.17%, and the proportion of the number of JD-specific SNP sites successfully detected in the number of all SNP sites in Table 1 is between 8.33% and 10.97%, determine that the sample to be tested is Jinding Duck.

[0010] The third aspect of the present invention provides a method for identifying a Fujian Mountain Shelduck population, comprising the following steps:

[0011] Extract the DNA of the sample to be tested; use the above-mentioned liquid-phase chip to test the sample to be tested, obtain the detection status of all SNP sites and count the number of successfully detected SNP sites among all SNP sites; when the proportion of the number of successfully detected SNP sites in the number of all SNP sites in Table 1 is between 66.83% and 73.17%, and the proportion of the number of FJ-specific SNP sites successfully detected in the number of all SNP sites in Table 1 is between 2.88% and 3.33%, determine that the sample to be tested is Fujian Mountain Shelduck.

[0012] The fourth aspect of the present invention provides a method for identifying a Shaoxing Duck population, comprising the following steps:

[0013] Extract the DNA of the sample to be tested; use the above-mentioned liquid-phase chip to test the sample to be tested, obtain the detection status of all SNP sites and count the number of successfully detected SNP sites among all SNP sites; when the proportion of the number of successfully detected SNP sites in the number of all SNP sites in Table 1 is between 66.83% and 73.17%, and the proportion of the number of SX-specific SNP sites successfully detected in the number of all SNP sites in Table 1 is between 16.22% and 17.33%, determine that the sample to be tested is Shaoxing Duck.

[0014] Further, the sample to be tested is the tissue or blood of the duck to be tested.

[0015] Further, the sample to be tested is the venous blood of the duck to be tested.

[0016] In summary, compared with the prior art, the present invention has the following beneficial advantages and effects:

[0017] (1) The present invention has mined a combination of SNP sites for duck breeding from large-scale sequencing data, a molecular probe set for detecting the combination of SNP sites, and a liquid-phase chip comprising the molecular probe set. This combination of SNP sites includes 1,021 unique SNP sites of the Jinding duck population, 367 unique SNP sites of the Fujian Sheldrake population, 1,612 unique SNP sites of the Shaoxing duck population, and 3,000 common SNP sites of the Jinding duck, Fujian Sheldrake, and Shaoxing duck populations. Moreover, a molecular probe set for detecting the combination of SNP sites and a liquid-phase chip comprising the molecular probe set have been developed. Using this combination of SNP sites or the molecular probe set, population screening, breeding, or identification of duck populations (Jinding duck, Fujian Sheldrake, and Shaoxing duck) can be carried out at the molecular level, with high specificity and accuracy. The liquid-phase chip provided by the present invention can accurately identify the three duck populations of Jinding duck, Fujian Sheldrake, or Shaoxing duck population, and has the advantages of simple operation, low cost, high efficiency, high detection throughput, etc., and is easy to be popularized and applied in laboratories and production practices, providing services for more scientific research institutions and enterprises.

[0018] (2) The present invention also provides methods for identifying the Jinding duck, Fujian Sheldrake, or Shaoxing duck population respectively, providing a new solution for the identification of these three duck populations. Compared with the existing identification methods, it can more quickly and accurately determine the population attribution of the Jinding duck, Fujian Sheldrake, or Shaoxing duck population. It helps to provide reliable experimental animal resources for scientific research personnel and improve the accuracy of research results. Detailed implementation manners

[0019] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments. In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0020] In the present invention: the unique SNP site refers to the SNP site found only in this breed; the common SNP site refers to the SNP site found in all three breeds (Jinding duck, Fujian Sheldrake, Shaoxing Sheldrake).

[0021] Duck breeding mainly relies on morphological differentiation, but there are problems such as poor accuracy, long selection cycle, and low efficiency. Molecular means provide new tools and methods for duck breeding, which helps to deeply understand the genetic characteristics of duck populations. SNP detection is an important means in genetic and medical research, so it is of great significance to mine a combination of SNP sites for duck breeding.

[0022] The present invention provides an application of a SNP locus combination, a molecular probe set for detecting the SNP locus combination, or a liquid chip containing the molecular probe set in duck breeding. Through whole-genome resequencing and analysis, the specific SNP loci and common SNP loci of Jinding ducks, Shaoxing ducks, and Fujian mountain mallard ducks were screened, and a SNP locus combination composed of specific SNP loci and common SNP loci was obtained. Based on this SNP locus combination, chip probe synthesis and performance determination were carried out, and finally the liquid chip was formulated, and a method for identifying Jinding ducks, Shaoxing ducks, or Fujian mountain mallard ducks was developed.

[0023] Example 1: Preparation of a liquid chip for duck breeding

[0024] The three duck breeds in the present invention are representatives of current Chinese SPF experimental breeding ducks and are raised in an isolated environment of the National Poultry Experimental Animal Resource Center (LARC). Among them, the Shaoxing ducks are from the Zhejiang Academy of Agricultural Sciences, the Jinding ducks are from the resource library of the National Waterfowl Base in Taizhou, Jiangsu, and the Fujian mountain mallard ducks are from the original breeding farm of Fujian mountain mallard ducks in Longyan City, Fujian. All ducks are isolated and raised in a positive pressure isolator using high-quality breeding eggs, and the duck flocks are regularly tested for pathogenic microorganisms, and positive individuals are eliminated to confirm that the duck flocks are not infected with Duck hepatitis A virus type I, Duck plague virus, Duck circovirus, Duck Tembusu virus, Goose parvovirus, Avian leukosis virus, Avian reovirus, Avian influenza virus, Newcastle disease virus, and Fowl adenovirus type III.

[0025] The preparation steps of the liquid chip are as follows:

[0026] S1. Blood sample collection: Using the wing vein method, collect blood samples of Jinding ducks, Shaoxing ducks, and Fujian mountain mallard ducks at 42 weeks of age. For each duck breed, 5 healthy females and 5 healthy males are selected respectively, and 10 blood samples are collected for each, totaling 30 blood samples.

[0027] S2. DNA extraction and library construction: Extract genomic DNA from whole blood samples. Use the NovaSeq6000 NGS platform to construct paired-end sequencing libraries for each individual, with an insert size of 300 bp to 400 bp, and perform quality control. The quality filtering criteria are as follows: 1) Remove reads with ≥10% unknown nucleotides; 2) Remove reads with >50% of the Phred quality score ≤20 bases; 3) Remove reads aligned with barcode adapters. Through this step, high-quality clean reads are obtained.

[0028] S3, SNP Identification and Filtering: Using the Burrows-Wheeler Aligner software, map the clean reads to the reference genome of ducks in the NCBI database to identify SNP sites. All samples are analyzed using the Unified Genotyper (GATK) in the Genome Analysis Toolkit. To reduce the false discovery rate, use VariantFiltration in GATK to filter SNPs, with the specific parameters: -Window 4, -filter "QD<2.0||FS>60.0||MQ<40.0," -G_filter "GQ<20". Use the software tool ANNOVAR to align and annotate the SNP sites to determine the physical location of each SNP site.

[0029] S4, High-quality SNP Screening: Obtain high-quality SNP sites according to the following criteria: 1) average coverage depth ≥5; 2) minor allele frequency ≥5%; 3) sample missing rate <10%.

[0030] S5, Genome-wide Selective Sweep Analysis: Use the R software package PopGenome to calculate the fixation index values in 100-kb sliding windows (step size = 10 kb) for genome-wide selective sweep analysis of different duck breeds. Then use the Z-transform (ZFST) to standardize the window-based FST values, and define the windows with ZFST values in the top 5% as selective regions, and the genes in these regions as candidate genes.

[0031] S6, Common Marker Screening: Set those with a depth less than 10× as missing, then filter out the sites with missing and intergenic sites, and select the sites that are consistent among all samples as the common marker sites for the three duck breeds of Jinding ducks, Shaoxing ducks, and Fujian mountain mallards.

[0032] S7, Specific Marker Screening: Set those with a depth less than 10× as missing, then filter out the sites with missing, and select the sites that are consistent within each population and not present in the other two populations as the specific markers for the Jinding duck, Shaoxing duck, or Fujian mountain mallard populations.

[0033] S8, SNP Locus Screening Results and Probe Design: Based on the proportion of the number of SNP loci detected on each chromosome, 12,000 SNP loci were selected as candidate loci, among which there were 6,000 common SNP loci and 6,000 unique SNP loci (2,000 for each population). Based on the SNP chromosome distribution, locus type distribution, and SNP density, probe design and evaluation were carried out on the selected loci. Loci with higher scores and a priority of 1 were selected, and the screening loci were determined according to the proportion of SNPs detected on each chromosome. Finally, a total of 3,000 common loci and 3,000 unique loci were determined (among them, there were 367 in Fujian Sheldrake Ducks, 1,021 in Jinding Ducks, and 1,612 in Shaoxing Sheldrake Ducks), obtaining an SNP locus combination containing 3,000 common loci and 3,000 unique loci. The specific information is shown in Table 1.

[0034] Table 1 Information of 6,000 SNP Loci in the SNP Locus Combination

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] The loci in the table are represented in the form of "chromosome number: specific locus on the chromosome: reference base / variant base of the locus"; JD-specific represents the SNP loci unique to Jinding ducks; FJ-specific represents the SNP loci unique to Fujian Shelducks; SX-specific represents the SNP loci unique to Shaoxing ducks.

[0068] S9. Through bioinformatics analysis, probes were designed and synthesized for the 6,000 selected loci, thereby obtaining a liquid-phase chip.

[0069] Example 2: Development and application of a duck population identification method

[0070] Using the liquid-phase chip prepared in Example 1, breed identification was performed on 30 Jinding ducks, 30 Shaoxing ducks, and 30 Fujian Shelducks. The specific steps are as follows:

[0071] (1) Sample collection: 2 mL of blood was collected through the wing vein.

[0072] (2) DNA extraction: The DNA in the collected blood was extracted using the phenol-chloroform extraction method.

[0073] (3) DNA quality detection: 1) Analyze and detect the integrity of DNA using agarose gel electrophoresis; 2) Detect the purity of the DNA sample to be tested using Nanodrop; 3) Accurately quantify the concentration of the DNA to be tested using Qubit.

[0074] (4) Liquid chip detection: Randomly fragment the qualified DNA sample to be tested with a length of about 200 bp by a disruptor. After end repair of the fragmented DNA, add an A tail, and then use a ligase to ligate the DNA fragment with an A tail to the sequencing adapter fragment. After probe hybridization, obtain the target region capture library and perform on-machine sequencing.

[0075] (5) Data processing and alignment analysis: Perform quality control filtering on the original data after sequencing by removing adapters and filtering low-quality data; use the BWA software to align the quality control filtered sequencing data to the genome (GCF_015476345.1_ZJU1.0_genomic), and then use the GATK software to analyze the SNP sites in the sequencing results to obtain the genotype typing results of SNP sites and exclude invalid SNP sites.

[0076] (6) Result interpretation and statistics: Statistically analyze the number of common SNP sites successfully detected, the number of JD-specific SNP sites, the number of FJ-specific SNP sites, and the number of SX-specific SNP sites to obtain the detection results shown in Table 2. The number of specific sites detected in Table 2 represents the number of specific sites detected for the corresponding variety.

[0077] Table 2 Detection results of the liquid chip

[0078]

[0079]

[0080]

[0081] Based on the proportion of the number of successfully detected sites accounting for 66.83% - 73.17% of all sites on the liquid chip, when the proportion of the number of JD-specific sites successfully detected in the range of 8.33% - 10.97% of all sites on the liquid chip, this variety is the Jinding duck;

[0082] Based on the proportion of the number of successfully detected sites accounting for 66.83% - 73.17% of all sites on the liquid chip, when the proportion of the number of SX-specific sites successfully detected in the range of 16.22% - 17.33% of all sites on the liquid chip, this variety is the Shaoxing duck;

[0083] On the basis that the proportion of the successfully detected loci in the total number of loci of the liquid chip ranges from 66.83% to 73.17%, when the proportion of the FJ-specific loci successfully detected in the total number of loci of the liquid chip ranges from 2.88% to 3.33%, this variety is the Fujian Sheldrake Duck.

[0084] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made to these embodiments by those skilled in the art once they learn the basic creative concept.

[0085] Obviously, those skilled in the art can make various changes and deformations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and deformations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and deformations.

Claims

1. Use of a SNP locus combination, a molecular probe set for detecting the SNP locus combination, or a liquid-phase chip containing the molecular probe set in duck breeding, characterized in that, The SNP locus combination contains 6,000 SNP loci; the physical positions of the SNP loci are determined based on alignment with the duck reference genome GCF_015476345.1_ZJU1.0_genomic; the specific information of the SNP loci is shown in the following table; The loci are represented in the form of "chromosome number: specific locus on the chromosome: reference base / variant base of the locus"; JD specific indicates SNP loci specific to Jinding ducks; FJ specific indicates SNP loci specific to Fujian Shelducks; SX specific indicates SNP loci specific to Shaoxing ducks; Common loci indicate loci shared by Jinding ducks, Fujian Shelducks, and Shaoxing Shelducks.

2. The application according to claim 1, wherein The duck breeding is duck species screening, duck species breeding, or duck species identification.

3. The application according to claim 1, characterized in that, The duck species is Jinding duck or Fujian Shelduck or Shaoxing duck.

4. A method for identifying a Jinding duck population, characterized in that It includes the following steps: Extract the DNA of the sample to be tested; Use the liquid-phase chip described in claim 1 to test the sample to be tested, obtain the detection status of all SNP loci, and count the number of successfully detected SNP loci among all SNP loci; When the proportion of the number of successfully detected SNP loci in all SNP loci is between 66.83% and 73.17%, and the proportion of the number of successfully detected JD-specific SNP loci in all SNP loci is between 8.33% and 10.97%, determine that the sample to be tested is a Jinding duck.

5. A method for identifying a Fujian Sheldrake duck population, characterized in that, It includes the following steps: Extract the DNA of the sample to be tested; Use the liquid-phase chip described in claim 1 to test the sample to be tested, obtain the detection status of all SNP loci, and count the number of successfully detected SNP loci among all SNP loci; When the proportion of the number of successfully detected SNP loci in all SNP loci is between 66.83% and 73.17%, and the proportion of the number of successfully detected FJ-specific SNP loci in all SNP loci is between 2.88% and 3.33%, determine that the sample to be tested is a Fujian Shelduck.

6. A method for identifying a Shaoxing duck population, characterized in that, It includes the following steps: Extract the DNA of the sample to be tested; Use the liquid-phase chip described in claim 1 to test the sample to be tested, obtain the detection status of all SNP loci, and count the number of successfully detected SNP loci among all SNP loci; When the proportion of the number of successfully detected SNP loci in all SNP loci is between 66.83% and 73.17%, and the proportion of the number of successfully detected SX-specific SNP loci in all SNP loci is between 16.22% and 17.33%, determine that the sample to be tested is a Shaoxing duck.

7. The identification method according to any one of claims 4 to 6, characterized in that The sample to be tested is the tissue of the duck to be tested or the blood of the duck to be tested.

8. The identification method according to claim 7, characterized in that, The sample to be tested is the blood of the duck to be tested.

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