SNP molecular marker combination for identifying white mountain goat bloodlines and application thereof

Abstract

The application discloses a SNP molecular marker combination for pedigree identification of Chongbai white goats, which is composed of 1000 (1K) SNP molecular markers, physical positions and numbers of the SNP molecular markers on a goat reference genome ARS1 are shown in Table 1 in the specification. By using the SNP molecular markers and based on a targeted capture chip genotyping technology, precise pedigree analysis of Chongbai white goat individuals can be realized, in particular, breed identification and kinship identification of Chongbai white goats. The application solves the technical problem that there is no suitable SNP chip detection means and analysis method for precise pedigree identification of Chongbai white goat individuals in the prior art.

Description

TECHNICAL FIELD

[0001] The present application relates to a SNP molecular marker combination for Chongming white goat pedigree identification and application thereof, and belongs to the technical field of biology. BACKGROUND

[0002] Single Nucleotide Polymorphism (SNP) molecular markers have a wide range of applications in agricultural breeding and other fields due to their large number, wide distribution, rich polymorphism, and ease of detection. However, the SNP density of such solid-phase chips is generally high, resulting in high typing costs.

[0003] Chongming white goat is one of the excellent local goat breeds in China, which is known for its tender meat, light odor, and strong adaptability. However, there is still a lack of specific SNP chip detection technology and methods for the pedigree tracing, kinship identification, and genetic diversity assessment of Chongming white goat individuals. Establishing a precise identification system suitable for local breeds has become a core issue that needs to be addressed for the protection of germplasm resources, breeding of superior varieties, and industrial development. SUMMARY

[0004] The present application aims to solve the above-mentioned problems of the prior art and provide a SNP molecular marker combination for Chongming white goat pedigree identification. By using this combination and based on the targeted capture chip genotyping technology, precise analysis of the pedigree of Chongming white goat individuals can be achieved.

[0005] The present application adopts the following technical solutions:

[0006] A SNP molecular marker combination for Chongming white goat pedigree identification, which is composed of 1000 (1K) SNP molecular markers. The physical location and number 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: 1000 SNP molecular marker positions of Shanghai Chongming white goat

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

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

[0015] A kit for identifying the pedigree of Chongbai goats, the kit comprising probes for detecting the above-mentioned combination of SNP molecular markers.

[0016] A SNP chip for identifying the pedigree of Chongbai goats, the SNP chip being provided with probes for detecting the above-mentioned combination of SNP molecular markers.

[0017] Further, the SNP chip is a liquid chip.

[0018] The above-mentioned SNP chip is used in the pedigree identification of Chongbai goats. The pedigree identification of Chongbai goats includes the breed identification of Chongbai goats.

[0019] The present application provides a combination of SNP molecular markers for identifying the pedigree of Chongbai goats, which is composed of 1000 1K SNP molecular markers. By using the combination of SNP molecular markers of the present application and based on the targeted capture chip genotyping technology, the pedigree of Chongbai goat individuals can be accurately analyzed. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 1 is a chromosome distribution map of Chongbai goat 1K breed identification SNP sites on the Chongbai goat reference genome ARS1. DETAILED DESCRIPTION

[0021] The technical solutions of the present application will be clearly and completely explained below in combination with the drawings and specific embodiments.

[0022] Example 1

[0023] 1. Screening of Chongbai goat 1K breed identification SNP sites

[0024] 1) Whole-genome resequencing of 60 individuals from the core conservation population of Shanghai Chongxian White goat (Shanghai Yuandu Sheep Farm) and 30 individuals from Jiangsu Haimen goat core conservation farm was performed. The raw reads were filtered using fastp software (0.20.0) with the following parameters: -n 10 -q 20 -u 40. The relevant whole-genome resequencing data has been stored in the publicly accessible website: http: / / www.cncb.ac.cn, with the biological project number PRJCA031876 and PRJCA031233, and the GSA number 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 from self-test, the genome resequencing data of three representative goats (Angora goat, Boer goat, Saanen dairy goat, Table 2) and five domestic representative goat breeds (Jining Green Goat, Matou Goat, Yunnan Mile Red-bone Goat, Guanzhong Dairy Goat, Yunshang Black Goat, Table 3) were downloaded from the National Center for Biotechnology Information (NCBI) public database (https: / / www.ncbi.nlm.nih.gov / ), totaling 232 individuals.

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

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

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[0032] 3) Through the variation identification and SNP screening of the whole genome data of the above-mentioned goat breeds, 1000 breed identification loci are obtained. The screening method is to filter and calculate the △MAF of each population at all loci under the condition that the deletion rate is less than 10% and the heterozygosity is less than 50%, that is, the absolute value of the minimum allele frequency (MAF) difference between the single population and other populations, and the 1K SNP loci with the highest △MAF of Chongbai goat (CM) breed are sorted, which can be understood as the SNP loci most representative of the breed information of Chongbai goat. The chromosome distribution map of these Chongbai goat 1K breed identification SNP loci on the goat reference genome ARS1 is shown in Figure 1 .

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

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

[0035] Example 2

[0036] Method for Chongbai goat pedigree identification:

[0037] 1. Extraction of goat DNA sample

[0038] From the anterior vena cava of the goat, blood (2-3 mL) is collected with a sterile syringe and injected into a blood collection tube to which an anticoagulant has been added in advance. The blood cells are separated from the plasma by centrifugation, and after washing the blood cell precipitate, a lysis buffer is added to release DNA and proteinase K is added to digest cell proteins. Acetic acid and isopropyl alcohol are added to coagulate and precipitate the DNA, and then the DNA precipitate is separated from the supernatant by centrifugation. The DNA precipitate is washed with acetate buffer to remove impurities and residual protease, and the DNA is dissolved in anhydrous solution or TE buffer, and stored at -20℃ for subsequent experiments (goat tissue samples (such as ear tissue or muscle tissue, etc.) can also replace goat blood DNA).

[0039] 2. Quality detection of DNA sample

[0040] The DNA concentration is determined by Agilent 2100 bioanalyzer (Agilent), and the integrity of the DNA is detected by 1% agarose gel electrophoresis. The qualified samples are stored in a 4℃ refrigerator for standby.

[0041] 3. Liquid chip detection

[0042] The liquid chip prepared in Example 1 was used to perform the liquid chip detection according to the standard procedure. The biotin-modified probes on the liquid chip can hybridize with the target region in the denatured resequencing library to form double-stranded DNA. Streptavidin-coated magnetic beads are used to adsorb the biotin-carrying molecules. After elution, amplification and targeted high-throughput deep sequencing, the obtained targeted sequencing data is subjected to quality control using fastp software (Chen et al., 2018). The sequencing data is then aligned to the goat reference genome ARS1 using BWA software (Li and Durbin, 2009). The standard procedure of GATK4 software (Van der Auwera et al., 2013) is used for detection, and the genotype data of 1K SNP sites of the detection sample is obtained.

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

[0044] The 1K SNP genotyping data file of the sample obtained by liquid chip detection is integrated with the Chongbai white goat breed identification model data set (10 breed identification model files, placed in the file download directory of the public management system of the Livestock and Poultry Genetic Resources Bank, and the download website link is http: / / 120.26.68.213:4569 / project1.html) established in Example 1 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 are extracted and combined according to the same 1K SNP sites, only the biallelic genotype sites are retained, and a New.bed file is generated. In the integrated New.bed file, the breed name of the sample to be detected is marked with “_”.

[0045] The integrated New.bed file is operated by using ADMIXTURE v1.3.0 software (Alexander et al., 2009). The ancestral cluster parameter K is set to 10 (representing a model set to 10 varieties), and the Admixture software is run under the Linux system with the following command: Admixture Tmp.bed 10-supervised-j12. The results after running (the file with the suffix Tmp.10.Q under the Dist folder) are imported into the Excel table, and the calculation results of the variety bloodline percentage composition of the corresponding samples to be detected are found (see https: / / dalexander.github.io / admixture / download.html for specific instructions of the ADMIXTURE software), that is, the bloodline component calculation of the sample to be detected is completed.

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

[0047] The identification method of Example 2 was used to analyze the genotypes of 1K SNP loci of 322 goats of 10 varieties described in Example 1, and the test results are shown in Table 4:

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

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[0064] Note: Column name is goat breed, model prediction results and consistency with the actual, row name is individual number, consistency TRUE indicates that the prediction results are consistent with the actual breed, the results show that 2 sample models are not consistent with the actual (FALSE), the rest are TRUE, the accuracy (consistency) is 320 / 322x100% = 99.38%.

[0065] As can be seen from Table 4, the 1K sites screened by the present application can accurately make precise identification of Chongbai white goat individuals, and the bloodline of other breeds is completely consistent with the actual breed (the model is determined as the individual of the breed according to the maximum percentage of the bloodline composition, and the verification result shows that the model judgment accuracy rate reaches 99.38%). The present application can not only qualitatively judge the bloodline of individuals, but also obtain the composition and percentage of the bloodline of each individual (see Table 4).

[0066] The SNP molecular marker combination of the present application lays a good foundation for the identification and screening of Chongbai white goat purebred individuals, at present, the kit designed based on the SNP molecular marker combination of the present application has been applied on a large scale in Shanghai Yundu White Goat Breeding Farm located in Chongming.

Claims

1. The application of SNP molecular marker combinations in the pedigree identification of Chongming white goats, characterized by: The SNP molecular marker combination consists of 1000 SNP molecular markers, and the SNP molecular markers' numbers and physical locations on the Chongming white goat reference genome ARS1 are shown in the table below: In the table above, the physical location of SNP molecular markers is represented by chromosome_physical location.

2. The application as described in claim 1, characterized in that, The pedigree identification of Chongming goats includes breed identification and kinship identification.

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