A specific molecular identification card for identifying the breed of Western Henan Black Pig and its application

Abstract

The present invention belongs to the field of pig breed identification, relates to the germplasm resources of Western Henan Black pigs, and particularly refers to a specific molecular identity card for identifying the breed of Western Henan Black pigs and its application. By studying the Queshan Black pig breed using SNP molecular markers, comprehensively analyzing with bioinformatics techniques, and preliminarily verifying the research results, a breed-specific molecular marker for Western Henan Black pigs is constructed, which can simply and clearly distinguish the differences between Western Henan Black pigs and other local pig breeds, can be used for the authenticity identification and genetic relationship analysis of Western Henan Black pig breeds, and provides an effective scientific basis for the intellectual property protection of Western Henan Black pig breeds. When identifying and evaluating the Western Henan Black pig breed, it is only necessary to compare and analyze the Western Henan Black pig breed with the existing specific molecular identity card to determine whether it is a Western Henan Black pig breed. This greatly reduces the time and cost of identifying and evaluating the germplasm resources of Western Henan Black pigs and improves the efficiency of identifying and evaluating the Western Henan Black pig breed.

Description

Technical Field

[0001] The present invention belongs to the field of pig breed identification, relates to the germplasm resources of Western Henan Black pigs, and particularly refers to a specific molecular identification card for identifying the breed of Western Henan Black pigs and its application. Background Art

[0002] Western Henan Black pigs are distributed in Sanmenxia City, Henan Province. They are bred from excellent local pigs: Nanyang Black pigs, Erhualian pigs, Laiwu pigs, and Western commercial pigs: Duroc pigs for generations, and have excellent characteristics such as strong reproductive ability, good meat quality, high disease resistance, and tolerance to roughage. In the process of breeding superior breeds, it is necessary to explore new technologies and means to provide guarantee for the cultivation of excellent breeds. How to quickly and efficiently identify and breed livestock and poultry varieties has become the key to the development and sustainable development of varieties in production. The effective protection and reasonable development of local varieties will contribute to the sustainable development of the pig industry in Henan Province and the enrichment of livestock resources diversity. In particular, the research on the specific genetic structure and characteristics of local pig breeds will help formulate protection plans for each breed according to the genetic situation of the breed, and promote the specific protection of local pig breeds. Preserving the unique variations, genes, and characteristics of each breed is extremely important for maintaining biodiversity and adapting to future environmental changes. Therefore, using bioinformatics technology to identify the unique genetic characteristics of local pig breeds in Henan is an important part of accurately protecting the germplasm genetic resources of local pigs.

[0003] With the development of sequencing technology, chip sequencing technology has become a powerful tool for high-throughput SNP genotyping. At the same time, when animals are subjected to long-term natural selection and artificial selection, corresponding genetic imprints will be left on their genomes. These genetic imprints are usually called selection signals. The research on selection signals is a research strategy based on the concept from genome to phenotype. Due to the lack of phenotypic records and small population size of local pig breeds in China, the analysis of livestock germplasm characteristics has increasingly become an important method. In order to achieve batch detection of Western Henan Black pigs, our research group has conducted long-term exploration. Summary of the Invention

[0004] To achieve the above object, the present invention provides a specific molecular identification card for identifying the breed of Western Henan Black pigs and its application.

[0005] The technical solution of the present invention is realized as follows:

[0006] A specific molecular identification card for identifying the breed of Western Henan Black pigs, wherein the SNP loci are a set of SNP loci with relatively high allele frequencies among Western Henan Black pig breeds.

[0007] Preferably, the SNP loci are located in the pig reference genome Ensembl Sscrofa version 11.1.

[0008] The set of the SNP sites includes the CNC10013894 site G / A, the CNC10014056 site C / T, the CNC10020115 site T / C, the CNC10020130 site A / C, the CNC10020514 site C / T, the CNC10023146 site, the CNC10023149 site, the CNC10031517 site, the CNC10031852 site, the CNC10032441 site, the CNC10042061 site, the CNC10042622 site, the CNC10050243 site, the CNC10050264 site, the CNC10050750 site, the CNC10050761 site, the CNC10061222 site, the CNC10061407 site, the CNC10062206 site, the CNC10062339 site, the CNC10062741 site, the CNC10070087 site, the CNC10071263 site, the CNC10081973 site, the CNC10082066 site, the CNC10090634 site, the CNC10090641 site, the CNC10090931 site, the CNC10091691 site, the CNC10111302 site, the CNC10130645 site, the CNC10133474 site, the CNC10134130 site, the CNC10140524 site, the CNC10140877 site, the CNC10151220 site, the CNC10151229 site, the CNC10151231 site, the CNC10152118 site and the CNC10171124 site.

[0009] The mutant types at the CNC10013894 locus are G / A, the mutant types at the CNC10014056 locus are C / T, the mutant types at the CNC10020115 locus are T / C, the mutant types at the CNC10020130 locus are A / C, the mutant types at the CNC10020514 locus are C / T, the mutant types at the CNC10023146 locus are C / T, the mutant types at the CNC10023149 locus are G / A, the mutant types at the CNC10031517 locus are T / C, the mutant types at the CNC10031852 locus are T / A, the mutant types at the CNC10032441 locus are C / T, the mutant types at the CNC10042061 locus are G / T, the mutant types at the CNC10042622 locus are C / T, the mutant types at the CNC10050243 locus are C / A, the mutant types at the CNC10050264 locus are T / C, the mutant types at the CNC10050750 locus are C / T, the mutant types at the CNC10050761 locus are T / C, the mutant types at the CNC10061222 locus are C / T, the mutant types at the CNC10061407 locus are G / A, the mutant types at the CNC10062206 locus are C / T, the mutant types at the CNC10062339 locus are A / G, the mutant types at the CNC10062741 locus are A / C, the mutant types at the CNC10070087 locus are A / G, the mutant types at the CNC10071263 locus are C / T, the mutant types at the CNC10081973 locus are T / C, the mutant types at the CNC10082066 locus are C / T, the mutant types at the CNC10090634 locus are G / A, the mutant types at the CNC10090641 locus are C / A, the mutant types at the CNC10090931 locus are A / C, the mutant types at the CNC10091691 locus are G / T, the mutant types at the CNC10111302 locus are C / T, the mutant types at the CNC10130645 locus are C / T, the mutant types at the CNC10133474 locus are C / T, the mutant types at the CNC10134130 locus are T / C, the mutant types at the CNC10140524 locus are A / G, the mutant types at the CNC10140877 locus are C / T, the mutant types at the CNC10151220 locus are A / G, the mutant types at the CNC10151229 locus are A / G, the mutant types at the CNC10151231 locus are A / G, the mutant types at the CNC10152118 locus are G / A and the mutant types at the CNC10171124 locus are T / C.

[0010] A gene chip for identifying the above specific molecular identity cards.

[0011] The application of the above gene chip in identifying the breed of Western Henan Black Pig.

[0012] Preferably, the steps are as follows:

[0013] (1) Collect tissue samples of the pigs to be tested and extract genomic DNA;

[0014] (2) Use a gene chip to perform SNP genotyping on the genomic DNA in step (1) to obtain genotype data of the pigs to be tested;

[0015] (3) Use PLINK software to merge the genotype data of the pigs to be tested with the genotypes of the SNP loci on the specific molecular identity card, and then perform principal component analysis.

[0016] Further, in step (1), the light absorption ratio of the genomic DNA at A260 / 280 is between 1.8 and 2.0, and the concentration is ≥50 ng / μL.

[0017] Further, in step (3), when the result of the principal component analysis of SNP genotyping is relatively close to the genetic distance of the Western Henan Black Pig population and clusters into a group, it is the Western Henan Black Pig.

[0018] The present invention has the following beneficial effects:

[0019] 1. Use PLINK software to merge the significant SNPs in group A and group B, and calculate the allele frequencies of each SNP in 11 breeds. Screen the SNP set with a higher allele frequency distribution in the experimental group than in the other 10 breeds as the specific molecular identity card of the Western Henan Black Pig breed.

[0020] 2. This patent combines the methods of genome-wide association analysis and selection signal analysis to determine the specific molecular identity card of the Western Henan Black Pig breed, laying a foundation for the development, utilization and identification of the Western Henan Black Pig breed. Through SNP molecular markers to study the Western Henan Black Pig breed, comprehensive analysis using bioinformatics technology, and preliminary verification of the research results, construct a breed-specific molecular marker for the Western Henan Black Pig, which can simply and clearly distinguish the differences between the Western Henan Black Pig and other local pig breeds, and can be used for the authenticity identification and genetic relationship analysis of the Western Henan Black Pig breed, providing an effective scientific basis for the intellectual property protection of the Western Henan Black Pig breed. When evaluating and identifying the Western Henan Black Pig breed, just compare and analyze the Western Henan Black Pig breed with the existing specific molecular identity card to determine whether it is the Western Henan Black Pig breed. This greatly reduces the time and cost of germplasm resource identification and evaluation of the Western Henan Black Pig, and improves the efficiency of Western Henan Black Pig breed identification and evaluation. Description of the Drawings

[0021] 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 for the description of the embodiments or the prior art. 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.

[0022] Figure 1 This is the Manhattan plot and QQ plot of the GWAS analysis results of the Western Henan Black Pig of the present invention.

[0023] Figure 2 This is the Manhattan plot of the selection signal analysis results of the Western Henan Black Pig of the present invention.

[0024] Figure 3 This is the principal component analysis verification plot of the breed-specific molecular identity card of the Western Henan Black Pig of the present invention. Detailed implementation manners

[0025] The following will, in combination with the embodiments of the present invention, clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention. Embodiment

[0026] A method for screening specific molecular identity cards of the germplasm resources of the Western Henan Black Pig, the steps are as follows:

[0027] (1) Ear sample collection

[0028] The experimental population is 1144 pigs, a total of 11 pig breeds, including 8 Chinese pig breeds: Western Henan Black Pig (n = 27, YX), Nanyang Black Pig (n = 10, NY), Huainan Pig (n = 10, HN), Yuenong Black Pig (n = 1036, YN), Queshan Black Pig (n = 10, QS), Laiwu Pig (n = 10, LWH), Erhualian Pig (n = 10, EHL), Min Pig (n = 6, MIN); 3 Western commercial pig breeds: Duroc (n = 10, DU), Large White Pig (n = 10, LW), Landrace (n = 5, LR). Use 75% alcohol to clean the ears of the pigs, use an ear sample clamp to cut a small amount of ear tissue, place it in a 2 mL centrifuge tube containing 75% alcohol, and store it in a -20°C refrigerator.

[0029] (2) Total DNA extraction, quality detection and genotyping

[0030] Use an animal tissue genomic DNA extraction kit to extract total DNA;

[0031] Using a DYY-6C electrophoresis apparatus, detection was performed by 1% agarose gel electrophoresis;

[0032] The concentration of DNA was detected using a Nanodrop-2000 ultraviolet spectrophotometer. Genomic DNA samples with an absorbance ratio (A260 / 280) between 1.8 and 2.0 and a concentration ≥ 50 ng / μL were used for whole-genome genotyping with the Illumina Porcine SNP50 BeadChip.

[0033] (3)Genotype data imputation and quality control

[0034] A total of 1,144 individuals and 51,315 SNPs were obtained through chip sequencing. The PLINK software was used to perform quality control on the chip data. The genotype data were filtered using the following parameters: individual genotype call rate (--mind) > 90%, marker genotype (--geno) call rate > 95%, minor allele frequency (--maf) > 1%, minimum Hardy-Weinberg equilibrium (--hwe) of 1.0E-6, located on autosomes. The Hidden Markov Model (HMM) algorithm was used to perform imputation of missing genotypes in the BEAGLE software.

[0035] (4)Screening SNP specific loci by genome-wide association study

[0036] The GEMMA software was used to perform genome-wide association analysis. The experimental group consisted of 27 YuXi black pigs (case), and the control group consisted of the remaining 10 breeds (control). The Bonferroni correction method was used to identify SNPs significantly associated with the breeds. The Manhattan plot of YuXi black pigs is shown in Figure 1 the left and the QQ plot is shown in Figure 1 the right. There are two threshold lines in the Manhattan plot. The threshold of the solid line is 0.05 / N (N is the number of chip loci used). The loci above the solid line are at the genome-wide significant level. The threshold of the dashed line is 1 / N. The loci above the dashed line are at the chromosome significant level. The closer the λ value in the QQ plot is to 1, the more reliable the results of the genome-wide association analysis; the Bonferroni correction method was used to identify SNPs significantly associated with the breeds. The set of these significant SNPs is group A.

[0037] (5)Selection signal analysis to screen SNP specific loci

[0038] Such as Figure 2As shown in the figure, the VCFtools software was used to calculate the genetic differentiation index (Fst), and the calculation method of taking the mean value with a sliding window was adopted. The specific parameters are as follows: the size of the sliding window (--fst-window-size) is 100,000 bp, and the step size of the sliding window (--fst-window-step) is 40,000 bp. The windows were sorted from large to small according to the Fst value, and the top 1% of the windows were defined as significant windows. Then, the PLINK software was used to extract the SNPs within the significant windows, and the set of these significant SNPs was group B.

[0039] (6)Allele frequency screening for SNP specific sites

[0040] The PLINK software was used to combine the significant SNPs of group A and group B, and the allele frequencies of each SNP in 11 breeds were calculated. The set of SNPs with higher allele frequencies in the experimental group than in the other 10 breeds was screened as the specific molecular identity card of the YuXi black pig breed.

[0041] Table 1 Specific molecular marker set of the YuXi black pig breed

[0042]

[0043] (7)The PLINK software was used to extract the above 40 SNPs of 11 breeds for principal component analysis verification.

[0044] (8)Application of the 40 SNP loci in identifying the YuXi black pig breed, and the SNP loci are located in the genome version EnsemblSscrofa 11.1.

[0045] Application example

[0046] A method for identifying a pig breed to be tested specifically includes the following steps:

[0047] 1. Extract the ear tissue samples of the pigs to be tested, extract the genomic DNA of the tissue samples, and genotype the above 40 loci through a chip. The genomic DNA is sent to Beijing Compson Biotechnology Co., Ltd. for SNP genotyping using the "Zhongxin No. 1" (Axiom) chip for indigenous pigs. The experimental principle of SNP genotyping by the chip is based on ligation reaction, in which two types of probes play a role. The first is the capture probe on the chip, which functions to fix the target DNA fragment to the chip surface. The second is the colorimetric probe, which is responsible for coloring the SNP chip (red and green fluorescence). The experiment is carried out in two rounds of hybridization. In the first round of hybridization, the target DNA hybridizes with the chip, and the capture probe grabs the matching target DNA fragment; the colorimetric probe hybridizes to the DNA fragment in the second round of hybridization. Then, using the recognition function of ligase, only the colorimetric probe complementary to the target DNA fragment will be ligated to the capture probe. Through fluorescence-labeled staining, SNP genotyping is performed under laser scanning to obtain the genotype data of the pigs to be tested.

[0048] 2. Use the PLINK software to combine the genotype data of the pigs to be tested with the genotype data of the above 11 breeds, and then perform principal component analysis and visualize the results using the R language. When the genetic distance between the pigs to be tested and the YuXi Black Pig population is relatively close and they cluster into a group, see Figure 3 , it can be determined that the pigs to be tested are YuXi Black Pigs.

[0049] Through the study of the YuXi Black Pig breed using SNP molecular markers, comprehensive analysis is carried out using bioinformatics technology, and the research results are preliminarily verified. By constructing the breed-specific molecular markers of the YuXi Black Pig, the differences between the YuXi Black Pig and other indigenous pig breeds can be clearly distinguished, which can be used for the authenticity identification and genetic relationship analysis of the YuXi Black Pig breed, providing an effective scientific basis for the intellectual property protection of the YuXi Black Pig breed. When evaluating and identifying the YuXi Black Pig breed, it is only necessary to compare and analyze the YuXi Black Pig breed with the existing specific molecular identity cards to determine whether it is the YuXi Black Pig breed. This greatly reduces the time and cost of germplasm resource evaluation and identification of the YuXi Black Pig and improves the efficiency of YuXi Black Pig breed evaluation and identification.

[0050] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for identifying the breed of Western Henan Black Pig, characterized in that: the method is achieved by identifying the following set of SNP loci; the SNP loci are located in the pig reference genome Ensembl Sscrofa version 11.1; the set of SNP loci is:

2. A gene chip for identifying the set of SNP loci, characterized in that, the SNP loci are located in the pig reference genome Ensembl Sscrofa version 11.1; the set of SNP loci is:

3. Use of the gene chip according to claim 2 in identifying the breed of Western Henan Black Pig.

4. According to the use described in claim 3, characterized in that, the steps are: (1) Collect tissue samples of the pig to be tested and extract genomic DNA; (2) Use the gene chip to perform SNP genotyping on the genomic DNA in step (1) to obtain the genotype data of the pig to be tested; (3) Use PLINK software to combine the genotype data of the pig to be tested with the genotype data of Western Henan Black Pig, and then perform principal component analysis.

5. According to the use described in claim 4, characterized in that: in step (1), the light absorption ratio of the genomic DNA at A260 / 280 is between 1.8 and 2.0, and the concentration is ≥50 ng / μL.

6. According to the use described in claim 5, characterized in that: when the result of the principal component analysis of SNP genotyping in step (3) has a relatively close genetic distance from the Western Henan Black Pig population and clusters into a group, it is a Western Henan Black Pig.

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