SNP (Single Nucleotide Polymorphism) molecular marker combination for identifying blood line of chongming white goat and application of SNP molecular marker combination

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker combination for identifying the bloodline of Chongming white goats, the SNP molecular marker combination consists of 1000 (1K) SNP molecular markers, and the physical positions and serial numbers of the SNP molecular markers on a goat reference genome ARS1 are shown in the specification table 1. By utilizing the SNP molecular markers and based on a targeted capture chip genotyping technology, accurate analysis of the blood lineage of the individual chongming white goat, especially variety identification and genetic relationship identification of the chongming white goat, can be realized. The technical problem that in the prior art, no appropriate SNP chip detection means and analysis method are available for accurate blood lineage identification of individual chongming white goats is solved.

Description

Technical Field

[0001] The invention relates to a SNP molecular marker combination for Chongbai goat pedigree identification and application thereof, belonging to the field of biotechnology. Background Art

[0002] Single nucleotide polymorphism (SNP) molecular markers are widely used in agricultural breeding and other fields due to their advantages such as large number, wide distribution, rich polymorphism and easy detection. However, the SNP density of such solid-phase chips is generally high, resulting in high typing costs.

[0003] Chongbai goat is one of the excellent local goat breeds in China. It is famous for its tender meat, light smell and strong adaptability. However, there is still a lack of targeted SNP chip detection technology and methods for pedigree tracing, kinship identification and genetic diversity assessment of Chongbai goats. Establishing an accurate identification system that is suitable for local breeds has become a core issue that needs to be broken through in germplasm resource protection, fine breed breeding and industrial development. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the above-mentioned prior art and to provide a SNP molecular marker combination for Chongbai goat pedigree identification. By using the SNP molecular marker combination and based on targeted capture chip genotyping technology, accurate pedigree analysis of Chongbai goat individuals can be achieved.

[0005] The present invention adopts the following technical solution:

[0006] A SNP molecular marker combination for Chongbai goat pedigree identification, the SNP molecular marker combination consisting of 1000 (1K) SNP molecular markers, the physical positions and numbers of the SNP molecular markers on the goat reference genome AR S1 (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_001704415.1 / ) are shown in Table 1:

[0007] Table 1 Locations of 1000 SNP molecular markers in Shanghai Chongbai goat

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[0013] Note: The physical position of SNP molecular markers is expressed as chromosome_physical position. For example, the physical position of the first SNP molecular marker is 1_2841644, which means it is at base 2841644 on chromosome 1 of the goat reference genome.

[0014] In the present invention, the SNP molecular markers are obtained by screening the genotype data of Chongbai goats and other 10 goat breeds at home and abroad. The screening method is to select the SNP sites with the highest absolute value (ΔMAF) of the difference in the minimum allele frequency (MAF) between the Chongbai goat breed (CM) and other breeds, and these sites can be understood as the most representative SNP sites (breed-informative SNPs) with the most information content of the Chongbai goat breed.

[0015] A kit for Chongbai goat pedigree identification, the kit comprising probes for detecting the above-mentioned SNP molecular marker combination.

[0016] A SNP chip for Chongbai goat pedigree identification, wherein the SNP chip is provided with probes for detecting the above-mentioned SNP molecular marker combination.

[0017] Furthermore, the SNP chip is a liquid phase chip.

[0018] The application of the SNP chip in Chongbai goat pedigree identification includes Chongbai goat breed identification.

[0019] Beneficial effects of the present invention: The present invention provides a SNP molecular marker combination for Chongbai goat pedigree identification, the SNP molecular marker combination is composed of 1000 1K SNP molecular markers, and the SNP molecular marker combination of the present invention and the targeted capture chip genotyping technology can be used to accurately analyze the pedigree of Chongbai goat individuals. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the chromosome distribution map of the SNP site identified for the Chongbai goat 1K variety in Example 1 on the Chongbai goat reference genome ARS1. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is clearly and completely described below in conjunction with the accompanying drawings and specific embodiments.

[0022] Example 1

[0023] 1. Screening of SNP loci for Chongbai goat 1K variety identification

[0024] 1) Whole genome resequencing was performed on 60 individuals from the core breeding population of Chongbai goats in Shanghai (Shanghai Yuandu Breeding Farm) and 30 individuals from the core breeding farm of Haimen goats in Jiangsu. Raw reads were filtered using fastp software (version 0.20.0) with parameters: -n 10 -q 20 -u 40). The relevant whole genome resequencing data have been deposited in a publicly accessible website: http: / / www.cncb.ac.cn, with the BioProject Nos. PRJCA031876 and PRJCA031233, and the GSA Nos. CRA020146 and CRA019905. Clean sequencing reads were then mapped to the goat reference genome ARS1 (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_001704415.1 / ) using Burrows-Wheeler Aligner (BWA) v0.7.17 (Li and Durbin, 2009). Genome Analysis Toolkit (GATK) v4.0 (Van der Auwera et al., 2013) was used for genetic variation mining (SNP calling).

[0025] 2) In addition to the above-mentioned 90 goat genome resequencing data, genome resequencing data of three representative goat breeds abroad (Angora goat, Boer goat, Saanen dairy goat, Table 2) and five representative goat breeds in China (Jining Blue Goat, Matou Goat, Yunnan Mile Red Bone Goat, Guanzhong Dairy Goat, Yunshang Black Goat, Table 3) were downloaded from the public database of the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / ), with a total of 232 individuals.

[0026] Table 2 Sources of whole genome resequencing data of three foreign goat breeds used in the present invention

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[0029] Table 3 Sources of whole genome resequencing data of five domestic goat breeds used in the present invention

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[0032] 3) Through the variation identification and SNP screening of the whole genome data of the above-mentioned goat breeds, 1,000 breed identification sites were obtained. The screening method is to filter according to the conditions of missing rate less than 10% and heterozygosity rate less than 50% and calculate the △MAF of each group at all sites, that is, the absolute value of the difference in the minimum allele frequency (MAF) between a single group and other groups, and take the 1K SNP sites with the highest △MAF ranking of the Chongbai goat (CM) breed. These sites can be understood as the most representative SNP sites (breed-informative SNPs) with the most information content of the Chongbai goat breed. The chromosome distribution map of these Chongbai goat 1K breed identification SNP sites on the goat reference genome ARS1 is shown in Figure 1 .

[0033] 2. Preparation of SNP liquid phase chip for Chongbai goat 1K pedigree identification

[0034] Based on the above-screened 1K breed identification SNP sites, according to the principle of DNA complementarity, one or more probes covering the sites are designed at each SNP site. The probe length is 110bp, the number of probes corresponding to the site is (1-2), the GC content of the probe is 30-70%, and the number of probe homologous regions is ≤5. The probes are synthesized by Shijiazhuang Boridi Biotechnology Co., Ltd., thereby producing a 1K SNP liquid phase chip for Chongbai white goat breed identification.

[0035] Example 2

[0036] Chongbai Goat Pedigree Identification Method:

[0037] 1. Extraction of goat DNA samples

[0038] Blood (2-3 mL) was collected from the anterior vena cava of goats using a sterile syringe and injected into a blood collection tube to which an anticoagulant had been added in advance. Blood cells were separated from plasma by a centrifugation step. After the blood cell precipitate was washed, a lysis buffer was added to destroy the cell membrane to release DNA, and proteinase K was added to digest the cell protein. Acetic acid and isopropanol were added to coagulate and precipitate the DNA, and then the DNA precipitate was separated from the supernatant by centrifugation. The DNA precipitate was washed with acetate buffer to remove impurities and residual proteases, and the DNA was dissolved with anhydrous solution or TE buffer and stored at -20°C for subsequent experiments (DNA from goat tissue samples (such as ear tissue or muscle tissue, etc.) can also replace goat blood DNA).

[0039] 2. DNA sample quality testing

[0040] The DNA concentration was determined using an Agilent 2100 bioanalyzer (Agilent), and the integrity of the DNA was detected using 1% agarose gel electrophoresis. The qualified samples were stored in a 4°C refrigerator for later use.

[0041] 3. Liquid chip detection

[0042] The liquid phase chip prepared in Example 1 was used and operated according to the standard liquid phase chip detection process. The probe modified with biotin on the liquid phase chip can hybridize with the target region in the denatured resequencing library to form a double strand, and the molecules carrying biotin are adsorbed by streptavidin-coated magnetic beads, and then eluted, amplified and targeted high-throughput deep resequencing is performed. The obtained targeted sequencing data was then quality controlled using fastp software (Chen et al., 2018), and the sequencing data was aligned to the goat reference genome ARS1 using BWA software (Liand Durbin, 2009), and the standard process of GATK4 software (Van derAuwera et al., 2013) was used to obtain the genotype data of the 1K SNP site of the test sample.

[0043] 4. Integration of sample data to be identified and model data

[0044] The obtained 1K SNP genotyping data file of the sample detected by the liquid chip was combined with the Chongbai goat breed identification model data set established in Example 1 (10 breed identification model files are placed in the file download directory in the public management system of the livestock and poultry genetic resources gene bank, and the download website link is http: / / 120.26.68.213:4569 / project1.html) using Plink v1.9 software (Purcell et al., 2007). The chip detection result data of the goat sample to be identified and the model files CM.ped and CM.map were extracted and merged according to the same 1K SNP site, only the di-allelic genotype sites were retained, and a New.bed file was generated. In the integrated New.bed file, the breed name of the sample to be detected was marked with "_".

[0045] The above integrated New.bed file was operated using ADMIXTURE v1.3.0 software (Alexander et al., 2009). The ancestral cluster parameter K was set to 10 (representing that the model was set to 10 varieties), and the Admixture software was run under the Linux system with the following command: Admixture Tmp.bed10–supervised-j12. The results after the operation (the file with the suffix Tmp.10.Q found in the Dist folder) were imported into the Excel table, and the calculation results of the pedigree percentage composition of the corresponding sample to be tested were found (for the specific instructions of ADMIXTURE software, see https: / / dalexander.github.io / admixture / download.html), which completed the calculation of the pedigree composition of the sample to be tested.

[0046] Prediction results and accuracy verification of Chongbai goat breed identification model:

[0047] The identification method of Example 2 was used to analyze the genotype of the 1K SNP site of 322 goats of the 10 breeds described in Example 1. The test results are shown in Table 4:

[0048] Table 4 Prediction results and accuracy verification of Chongbai goat breed identification model

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[0064] Note: The column names are goat breed, model prediction results and consistency with the actual results. The row names are individual numbers. TRUE in the consistency indicates that the prediction results are consistent with the actual breed. The results show that there are 2 samples where the model predictions are inconsistent with the actual results (FALSE), and the rest are TRUE. The accuracy (consistency) is 320 / 322×100%=99.38%.

[0065] As can be seen from Table 4, the 1K locus screened by the present invention can accurately identify the Chongbai goat individuals, and the pedigree judgments of other breeds are completely consistent with the pedigrees of the actual breeds (the model determines the individual of the breed based on the maximum percentage of the pedigree components, and the verification results show that the model judgment accuracy rate reaches 99.38%). The present invention can not only make qualitative judgments on the pedigree of individuals, but also calculate the composition and percentage of the pedigree of each individual (see Table 4).

[0066] The SNP molecular marker combination of the present invention has laid a good foundation for the identification and screening of purebred Chongming white goats. At present, the kit designed based on the SNP molecular marker combination of the present invention has been widely used in the Shanghai Yuandu White Goat Conservation Farm located in Chongming.

Claims

1. A SNP molecular marker combination for Chongbai goat pedigree identification, characterized in that: The SNP molecular marker combination consists of 1000 SNP molecular markers, and the physical positions and numbers of the SNP molecular markers on the Chongbai goat reference genome ARS1 are shown in Table 1 of the specification.

2. A kit for Chongbai goat pedigree identification, characterized in that: The kit comprises probes for detecting the SNP molecular marker combination according to claim 1.

3. A SNP chip for Chongbai goat pedigree identification, characterized in that: The SNP chip is provided with probes for detecting the SNP molecular marker combination according to claim 1.

4. The SNP chip for Chongbai goat pedigree identification as claimed in claim 3, characterized in that: The SNP chip is a liquid phase chip.

5. Use of the SNP chip according to claim 3 or 4 in pedigree identification of Chongbai goats.

6. The use according to claim 5, characterized in that The Chongbai goat pedigree identification includes the Chongbai goat breed identification and kinship identification.

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