A SNP molecular marker combination, chip and preparation method and application thereof for Beijing black pig genotyping

Abstract

The invention relates to the fields of molecular biology, functional genomics, bioinformatics, and genomic breeding technology, and specifically discloses a SNP molecular marker combination, a chip, and a preparation method and application thereof for Beijing black pig genotyping. The invention relates to a SNP molecular marker combination for Beijing black pig genotyping, which is composed of 49,963 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NO: 1-49,963. The molecular marker combination can be used to prepare a Beijing black pig molecular breeding chip, and can be further applied to the specific identification of Beijing black pigs and other pig breeds (European commercial pig breeds, Chinese local pig breeds), target trait QTL, associated sites and candidate gene identification, kinship identification, and family division.

Description

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 202210433080.4 filed on April 22, 2022, entitled “A SNP molecular marker combination, chip, preparation method and application thereof for Beijing black pig genotyping”, all disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the technical fields of genomics, bioinformatics, molecular biology and genome breeding, and in particular to a SNP molecular marker combination, a chip and a preparation method and application thereof for Beijing black pig gene typing. Background Art

[0004] Beijing Black Pig is bred by crossbreeding large numbers of offspring from foreign pig breeds such as Large White Pig, Berkshire Pig, Soviet Large White Pig and Caucasian Pig with local pig breeds. The breed was officially named "Beijing Black Pig" in 1982 and was later designated as a maternal stock pig. Beijing Black Pig combines the excellent characteristics of domestic and foreign pig breeds, combining the characteristics of pig breeds such as tolerance to roughage, early maturity and high productivity, and tender meat with the characteristics of large body, fast growth and high meat content.

[0005] However, the current core group size of Beijing Black pigs is too small to implement effective breeding and selection, and the group production performance needs to be further improved. Developing a specialized chip for Beijing Black pig molecular breeding, realizing the rational development and utilization of Beijing Black pig specialty varieties, cultivating specialized Beijing Black pig excellent varieties and high-quality pig matching systems, developing Beijing Black pig germplasm resources, and developing high-quality and high-end pork products based on this variety are of great significance to accelerate the selection and improvement of Beijing Black pigs and promote the development of specialty animal husbandry.

[0006] SNP refers to the variation of a single nucleotide at the genome level, including molecular markers formed by the deletion, insertion, conversion and transversion of a single base, which are characterized by large number and wide distribution. As a genetic marker, SNP contributes to the genetic variation of complex traits, and is therefore widely used in genetic research. The SNP chip is made by fixing a fluorescently labeled DNA probe on a silicon wafer, and then performing SNP typing by hybridizing the probe DNA with the genomic DNA. SNP binds to the probe on the surface of the silicon wafer instead of the genomic sequence, so that the DNA of a large number of individuals can be fixed on a chip for analysis.

[0007] Illumina's Infinium SNP chip technology is currently a relatively mature and widely used whole-genome SNP detection platform. First, the denatured DNA is hybridized with the probes on the chip microbeads, then the unhybridized DNA is removed, and the obtained DNA is subjected to an extension reaction of specially labeled bases; finally, the SNP information is converted into visible fluorescent information through the immunological combination of the label and the fluorescent group.

[0008] At present, mature commercial SNP chips for pigs are mainly based on the PorcineSNP60 chip developed by Geneseek based on the Illumina platform, the GGP-Porcine HD chip, and the Compass PorcineSNP55 chip designed by Compson. Another platform is the Affymetrix platform. Currently, the main pig SNP chips are high-density chips developed by Affymetrix, which contain about 650,000 SNPs, but are expensive and rarely used.

[0009] However, the current commercial chips have certain limitations due to the source of SNP sites, and most of them are designed for foreign pig breeds such as Large White Pig, Landrace Pig, Duroc, etc., and have poor adaptability to the Beijing Black Pig. Therefore, it is necessary to develop targeted chips suitable for Beijing Black Pig. Summary of the invention

[0010] The purpose of the present invention is to provide a SNP molecular marker combination for Beijing black pig genotyping, a chip and a preparation method and application thereof.

[0011] Specifically, the technical solution of the present invention is as follows:

[0012] In a first aspect, the present invention provides a SNP molecular marker combination for Beijing black pig genotyping, which consists of 49,963 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-49,963, respectively.

[0013] In a second aspect, the present invention provides the use of the above-mentioned SNP molecular marker combination in the preparation of a Beijing black pig molecular breeding chip.

[0014] In a third aspect, the present invention provides a Beijing black pig molecular breeding 50K SNP chip, which comprises 1-49,963 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-49,963, respectively.

[0015] The above-mentioned SNP molecular marker combination for Beijing black pig genotyping or Beijing black pig molecular breeding 50K SNP chip, wherein the SNP site of the SNP molecular marker is located at the 71st position of the nucleotide sequence shown in SEQ ID NO.1-49,963.

[0016] The present invention provides a 50K SNP chip for molecular breeding of Beijing black pigs, wherein the 71st base of each sequence is a SNP site. The SNP molecular markers mainly come from five types of SNP sites: the first type is derived from sites with good polymorphism and high detection rate in the Beijing black pig population of the Zhongxin No. 1 PLUS pig breeding chip, including 23,299 SNP sites; the second type is derived from sites (preferentially exon regions and regulatory regions) significantly associated with existing important economic traits (especially meat quality traits) reported in existing literature and public databases, including 937 SNP sites; the third type is derived from sites with large absolute allele frequency differences in the results of SNP variation detection of Beijing black pigs and commercial pig breeds, including 2,536 SNP sites; the fourth type is derived from sites significantly associated with meat quality traits in the results of Beijing black pig whole genome association analysis, including 31 SNP sites; the fifth type is derived from sites detected by SNP variation detection of Beijing black pig resequencing data, including 23,160 SNP sites. The above five types of probes contain a total of 49,963 SNP sites.

[0017] In a fourth aspect, the present invention provides a method for preparing the Beijing Black Pig Molecular Breeding 50K SNP chip, which comprises the following steps:

[0018] Step ①: Obtaining the first type of probe: Use The Beijing black pig population was tested with the Zhongxin No. 1 PLUS pig breeding chip (available from Beijing Compson Biotechnology Co., Ltd.), and the chip data was quality controlled. The quality control standards were: SNP detection rate < 95%, minimum allele frequency < 0.1, Hardy-Weinberg equilibrium test P value < 10 -6 And SNP sites without chromosome location information; the location information of SMIC-1 PLUS pig breeding chip was converted from Sus scrofa 10.2 version to Susscrofa 11.1 version reference genome, and the first category of candidate SNPs was obtained;

[0019] Step ②: Obtaining the second type of probe: Combine the public database NCBI and QTLdatabase to obtain the internal and upstream and downstream SNP sites of candidate genes related to pork quality traits, growth traits, reproductive traits and health traits, genome-wide association analysis and QTL results significant SNPs, and based on the Beijing Black Pig resequencing data, remove sites with MAF < 0.05 to obtain the second type of candidate SNPs;

[0020] Step ③: Obtaining the third type of probe: Based on the resequencing data of Beijing black pigs, large white pigs and landrace pigs, the sites with absolute allele frequency differences of Beijing black pigs, large white pigs and landrace pigs greater than or equal to 0.6 were screened, and based on the resequencing data of Beijing black pigs, the sites with MAF < 0.05 were eliminated to obtain the third type of candidate SNPs;

[0021] Step ④: Acquisition of the fourth type of probe: Use the Zhongxin No. 1 PLUS pig breeding chip to detect the Beijing black pig population, and conduct a whole-genome association analysis based on the chip data and meat quality phenotype data of the Beijing black pig. The meat quality phenotype data include intramuscular fat, protein content, moisture content, collagen content, drip loss, meat color and pH value 24 hours after slaughter (a total of 7 traits); the loci significantly associated with meat quality traits identified by the whole-genome association analysis are used as the fourth type of candidate SNPs;

[0022] Step ⑤: Obtaining the fifth type of probe: Based on the Beijing Black Pig resequencing data, retain the SNPs with MAF ≥ 0.1 to obtain the fifth type of candidate SNPs;

[0023] Step ⑥: In order to evenly distribute SNP sites at the whole genome level, the autosomes and X chromosomes were divided into 48 kb windows, and one SNP was selected for filling in each window according to the priority of the first to fifth categories of probes;

[0024] Step 7: The selected SNP sites are scored by the Infmium iSelect scoring system (http: / / www.illumina.com / ), and the sites with intragenic scores < 0.7 and intergenic scores < 0.9 are removed; for the deleted unqualified SNP sites, the nearest SNP sites are selected to supplement them, and the scores are performed again until all sites are qualified, and then the obtained tag sequences are used for the preparation of SNP chips.

[0025] In the present invention, the Infmium chip manufacturing technology is used to manufacture the SNP chip. Specifically, the obtained tag sequence can be handed over to Illumina to design and manufacture the Infmium SNP chip.

[0026] In a fifth aspect, the present invention provides the use of the above-mentioned SNP molecular marker combination for Beijing black pig genotyping or the Beijing black pig molecular breeding 50K SNP chip in Beijing black pig whole genome association analysis.

[0027] In the whole genome association analysis of Beijing black pig, the present invention uses the above-mentioned Beijing black pig molecular breeding 50KSNP chip to perform genotype detection on Beijing black pig, and determines the relevant phenotypic traits, and performs whole genome association analysis based on the linear mixed model. The expression of the linear mixed model is:

[0028] y=Xα+Yβ+Zγ+e

[0029] y is the phenotype vector, α is the SNP marker effect vector, X is the association matrix corresponding to α, coded as 0, 1, 2, β is the non-genetic fixed effect vector, Y is the association matrix corresponding to β, and γ is the residual polygenic effect vector, which follows a normal distribution G is the genomic kinship matrix, is the additive genetic variance, Z is the correlation matrix corresponding to γ, e is the residual vector, and obeys I is the identity matrix, are random residuals.

[0030] In a sixth aspect, the present invention provides the use of the above-mentioned SNP molecular marker combination for Beijing black pig genotyping or the Beijing black pig molecular breeding 50K SNP chip in Beijing black pig variety identification.

[0031] When identifying an unknown breed of pig, the present invention uses the Beijing black pig molecular breeding 50K SNP chip to perform genotype detection on the unknown breed of pig, merges the genotype data with the chip data of the known Beijing black pig, uses PLINK software to quality control the chip data and calculate the IBS distance matrix between individuals, and based on the IBS distance matrix, uses the neighbor-joining method of PHYLIP software to construct a phylogenetic tree, and finally uses Figtree software to visualize the phylogenetic tree to determine whether the unknown breed of pig is a Beijing black pig, thereby performing breed identification.

[0032] The quality control criteria were as follows: ① individual genotype frequency > 0.95; ② SNP call rate > 0.95; ③ minimum allele frequency (MAF) > 0.05; ④ Hardy-Weinberg equilibrium test P value > 10 -6 .

[0033] The chip of the present invention can be used to identify the varieties of Beijing black pigs and other pig breeds (European commercial pig breeds and Chinese local pig breeds).

[0034] In a seventh aspect, the present invention provides the use of the above-mentioned SNP molecular marker combination for Beijing black pig genotyping or the Beijing black pig molecular breeding 50K SNP chip in Beijing black pig breeding.

[0035] The beneficial effects of the present invention are at least:

[0036] The Beijing Black Pig molecular breeding specialized SNP chip of the present invention is designed in accordance with its own needs. The source of SNP sites is more consistent with the characteristics of the population, and the chip density is reduced by selecting the effective number of SNPs, thereby saving costs to the greatest extent. The chip detection fee of the present invention is 150 yuan / chip, which is 50 yuan lower than the cost of the SMIC No. 1 PLUS pig breeding chip.

[0037] In addition, compared with the current mainstream 50k SNP chip for pigs, the SMIC-1 PLUS pig breeding chip, the chip made with the SNP molecular marker combination of the present invention has a more uniform distribution of sites: when the position information of the SMIC-1 PLUS pig breeding chip is converted from Susscrofa 10.2 version to the Sus scrofa 11.1 version reference genome, the standard deviation of the distance between adjacent SNPs is 50.4kb, indicating that the discrete degree of the distance between adjacent SNPs is large; while the standard deviation of the distance between adjacent SNPs of the chip of the present invention is 9.4kb, which is much lower than that of the SMIC-1 PLUS pig breeding chip, indicating that the discrete degree of the distance between adjacent SNPs is smaller and the distribution in the whole genome is more uniform.

[0038] From the perspective of polymorphism, the SNP sites of the chip of the present invention have better polymorphism in the Beijing black pig population. The average minimum allele frequency of the SMIC-1 PLUS pig breeding chip in the Beijing black pig population is 0.220, while that of the chip of the present invention is 0.276.

[0039] The SNP molecular marker combination provided by the present invention has the characteristics of uniform distribution of SNP sites and high whole-genome coverage. The use of these SNP markers for whole-genome breeding can effectively improve the degree of association with traits through linkage disequilibrium, thereby ensuring the accuracy of breeding value estimation. The present invention is based on the resequencing data of Beijing black pigs when screening sites. The selected sites have high polymorphism in the Beijing black pig population, ensuring the high quality of the chip sites. This chip adds significant SNP sites related to pig economic traits (especially meat quality traits), thereby improving the practicality of the chip. The present invention uses the resequencing data of Beijing black pigs and commercial pig breeds when screening SNP sites, and screens out sites with large differences in absolute allele frequencies between breeds, which can be used for Beijing black pig breed identification, and is conducive to protecting Beijing black pig germplasm resources. In addition, this chip can also be used for Beijing black pig whole-genome selection breeding, Beijing black pig target trait QTL, associated sites and candidate gene identification, kinship identification, family division, etc., and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a frequency distribution histogram of the minimum allele frequency (MAF) of the SNP site in Example 1 of the present invention.

[0041] Figure 2It is a box plot of the distance between adjacent SNP sites in Example 1 of the present invention (unit: kb), the ordinate is the SNP interval (Interval), and the abscissa is the chromosome (CHR) number.

[0042] Figure 3 This is a Manhattan plot of the genome-wide association analysis results of Beijing black pig intramuscular fat traits in Example 2 of the present invention. The ordinate is the p-value of each SNP site, which is -log10 transformed, and the abscissa is the chromosome number.

[0043] Figure 4 This is a phylogenetic tree diagram of Beijing Black Pig, European commercial pig breeds, and Chinese local pig breeds in Example 3 of the present invention. The abbreviations represent breed information, and the digital numbers represent the individual numbers of each breed. BJB represents Beijing Black Pig, DU represents Duroc Pig, LW represents Large White Pig, LD represents Landrace Pig, MS represents Horse Body Pig, HN represents Huainan Pig, LT represents Lantang Pig, LC represents Luchuan Pig, and MIN represents Min Pig. DETAILED DESCRIPTION

[0044] The preferred embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention. If not otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions. If not otherwise specified, the equipment and reagents used in each example are all conventionally commercially available.

[0045] Example 1 Preparation method of 50K SNP chip for molecular breeding of Beijing black pig

[0046] SNP site screening process: In order to evenly distribute SNP sites at the whole genome level, the autosomes and X chromosomes were divided into 48kb windows, and each window was filled with 1 SNP according to the priority of the first to fifth categories of probes.

[0047] 1. Acquisition of the first type of probe:

[0048] The Beijing black pig population was tested using the SMIC-1 PLUS pig breeding chip, and the chip data was quality controlled. The quality control standards were: SNP detection rate < 95%, minimum allele frequency < 0.1, Hardy-Weinberg equilibrium test P value < 10 -6 And SNP sites without chromosome location information.

[0049] The position information of the SMIC-1 PLUS pig breeding chip is the Sus scrofa 10.2 (https: / / www.ncbi.nlm.nih.gov / assembly / GCF_000003025.5 / ) reference genome. The position information was converted to the Sus scrofa 11.1 (https: / / www.ncbi.nlm.nih.gov / assembly / GCF_000003025.6 / ) reference genome through the UCSC online website (http: / / genome.ucsc.edu / cgi-bin / hgLiftOver) to obtain the first category of candidate SNPs.

[0050] The length of the whole genome of a pig (excluding the Y chromosome) is about 2.4Gb. In order to ensure that these SNPs are distributed as evenly as possible at the whole genome level, the present invention divides the whole length of the pig genome into a window of 48kb each. The specific method for determining SNPs: 1) If there are more than or equal to 2 alternative SNPs in the window, select the SNP closest to the midpoint of the window; 2) If the number of alternative SNPs in the window is 1, the alternative SNP directly becomes the final target SNP; 3) If there is no alternative SNP of the first type of probe in the window, the window is retained and waits for the alternative SNP of the second type of probe to be filled. 4) Set the filtering standard of 20k≤the spacing between adjacent SNPs / the window spacing≤75k, and remove SNPs that do not meet this standard. Finally, 23,299 SNPs of the first type of probe were determined.

[0051] 2. Acquisition of the second type of probe:

[0052] Combined with the public database NCBI (https: / / www.ncbi.nlm.nih.gov / pubmed / ) and QTLdatabase (https: / / www.animalgenome.org / cgi-bin / QTLdb / index / ), we obtained the internal and upstream and downstream SNP sites (exponential regions and regulatory regions) of candidate genes related to economic traits such as pork quality traits, growth traits, reproductive traits and health shape, as well as genome-wide association analysis and QTL results. Based on the Beijing Black pig resequencing data, we eliminated sites with MAF < 0.05 to obtain the second category of candidate SNPs.

[0053] After completing the screening of the first type of probes, for windows without SNPs, fill in the second type of probes. The specific method is as follows: 1) If there are more than or equal to 2 candidate SNPs in the window, select the SNP closest to the midpoint of the window; 2) If the number of candidate SNPs in the window is 1, the candidate SNP directly becomes the final target SNP; 3) If there is no candidate SNP of the second type of probe in the window, keep the window and wait for the candidate SNP of the third type of probe to be filled. 4) Set the filtering standard of 20k≤the spacing between adjacent SNPs / the window spacing≤75k, and remove the SNPs that do not meet this standard. Finally, 937 SNPs of the second type of probes were determined.

[0054] 3. Obtaining the third type of probe:

[0055] Based on the resequencing data of Beijing Black Pig, Large White Pig and Landrace Pig, the loci with absolute allele frequency differences of Beijing Black Pig, Large White Pig and Landrace Pig greater than or equal to 0.6 were screened. Based on the resequencing data of Beijing Black Pig, the loci with MAF < 0.05 were eliminated to obtain the third category of candidate SNPs.

[0056] After completing the screening of the second type of probes, for windows without SNPs, fill in the third type of probes. The specific method is as follows: 1) If there are more than or equal to 2 candidate SNPs in the window, select the SNP closest to the midpoint of the window; 2) If the number of candidate SNPs in the window is 1, the candidate SNP directly becomes the final target SNP; 3) If there is no candidate SNP of the third type of probe in the window, keep the window and wait for the candidate SNP of the fourth type of probe to be filled. 4) Set the filtering standard of 20k≤the spacing between adjacent SNPs / the window interval≤75k, and remove the SNPs that do not meet this standard. Finally, 2,536 SNPs of the third type of probes were determined.

[0057] 4. Acquisition of the fourth type of probe:

[0058] A genome-wide association analysis was performed based on the chip data and meat quality phenotype data of Beijing Black Pig. The meat quality phenotype data included seven traits: intramuscular fat, protein content, moisture content, collagen content, drip loss, meat color, and pH 24 hours after slaughter. All loci significantly associated with meat quality traits identified by the genome-wide association analysis were retained as the fourth category probes. Finally, 31 SNPs of the fourth category probes were determined.

[0059] 5. Acquisition of the fifth type of probe:

[0060] Based on the Beijing Black Pig resequencing data, SNPs with MAF ≥ 0.1 were retained to obtain the fifth category of candidate SNPs.

[0061] For windows without SNPs, fill in the fifth type of probes. The specific method is as follows: 1) If there are more than or equal to 2 candidate SNPs in the window, select the SNP closest to the midpoint of the window; 2) If there is only one SNP in the window, then the SNP directly becomes the target SNP. 3) If there is no candidate SNP for the fifth type of probe in the window, relax the standard and use SNPs with MAF ≥ 0.05 for filling.

[0062] Finally, based on the Beijing Black pig resequencing data, SNPs with MAF ≥ 0.05 located in the non-homologous region of the Y chromosome were selected on the basis of uniformity, and a total of 15 SNPs were selected. These SNPs are only used to identify the sex of Beijing Black pigs when the sex information of the phenotype is unknown.

[0063] In summary, 23,160 SNPs of the fifth category probes were finally determined.

[0064] The screened SNP sites were submitted to Illumina for scoring by the Infmium iSelect scoring system (http: / / www.illumina.com / ), and sites with intragenic scores <0.7 and intergenic scores <0.9 were removed. For the deleted unqualified SNP sites, the nearest SNP sites were selected for supplementation and scored again. According to the above steps, 49,963 SNP sites were identified and screened, and finally a total of 49,963 SNP sites were obtained. According to the design requirements of Illumina Infmium iSelect HD, 49,963 beads are required. All 49,963 SNP sites are 49,963 DNA sequences shown in SEQ ID NO: 1-49,963. These tag sequences were submitted to Illumina for design and production of the Infmium SNP chip. The number of chip sites distributed on each chromosome is shown in Table 1.

[0065] This 50K SNP chip has three advantages: first, it has functional relevance to economic traits. The chip collects and identifies a large number of significant loci related to pork quality traits, growth traits, reproductive traits, and health traits, which increases the practicality of the chip; second, the selection of loci is based on the Beijing Black Pig resequencing data, ensuring that the selected loci have a high degree of polymorphism (see the MAF distribution of SNP loci for details). Figure 1 ), and the sites are evenly distributed (see the distance between adjacent SNP sites Figure 2 ), ensuring the accuracy of breeding value estimation; thirdly, it is specific. The chip loci are specially designed for the Beijing Black Pig breed, and the loci that are significantly different from those of commercial pig breeds are selected, which can specifically identify the Beijing Black Pig from other pig breeds.

[0066] Table 1 Distribution of chip loci on each chromosome

[0067]

[0068] Example 2 Application of 50K SNP chip in genome-wide association analysis for molecular breeding of Beijing black pigs

[0069] The Beijing black pig molecular breeding 50K SNP chip provided in Example 1 of the present invention was used to perform genotyping on the Beijing black pig to obtain genotyping data. The intramuscular fat trait and the carcass weight trait of the Beijing black pig were measured to obtain phenotypic data.

[0070] Beagle software was used to fill in the chip data, and then PLINK software was used to perform quality control. The quality control criteria were as follows: ① individual genotype frequency > 0.95; ② SNP call rate > 0.95; ③ minimum allele frequency (MAF) > 0.05; ④ Hardy-Weinberg equilibrium test P value > 10 -6 ; The SNP site information obtained after quality control is used for subsequent whole-genome association analysis.

[0071] The quartile test method was used to eliminate outliers for intramuscular fat traits and carcass weight phenotypic traits.

[0072] GEMMA software was used to perform genome-wide association analysis based on a linear mixed model. The linear mixed model was expressed as:

[0073] y=Xα+Yβ+Zγ+e

[0074] y is the intramuscular fat phenotype vector, α is the SNP marker effect vector, X is the association matrix corresponding to α, coded as 0, 1, 2, β is the non-genetic fixed effect vector, including population mean, sex, and carcass weight, Y is the association matrix corresponding to β, and γ is the residual polygenic effect vector, which follows a normal distribution G is the genomic kinship matrix, is the additive genetic variance, Z is the correlation matrix corresponding to γ, e is the residual vector, and obeys I is the identity matrix, are random residuals.

[0075] The significance test was performed using the Wald test, with p < 1 × 10 -5 As a threshold for potential genome-wide significance, the Manhattan result obtained is Figure 3 The results showed that the genome-wide significant association site located on chromosome 1 may be a regulatory site for the trait of intramuscular fat.

[0076] Example 3: Beijing Black Pig Molecular Breeding 50K SNP Chip for Variety Identification

[0077] The Beijing black pig molecular breeding 50K SNP chip provided in Example 1 of the present invention was used to perform genotype detection on DNA extracted from 90 pigs of 9 breeds, including 22 Beijing black pigs (BJB), 22 Duroc pigs (DU), 16 Large White pigs (LW), 5 Landrace pigs (LD), 5 Horse Body pigs (MS), 5 Huainan pigs (HN), 5 Lantang pigs (LT), 5 Luchuan pigs (LC) and 5 Min pigs (MIN).

[0078] PLINK software was used to perform quality control on the chip data. The quality control criteria were as follows: ① individual genotype frequency > 0.95; ② SNP call rate > 0.95; ③ minimum allele frequency (MAF) > 0.05; ④ Hardy-Weinberg equilibrium test P value > 10 -6 ; The SNP site information obtained after quality control is used for subsequent variety identification and analysis.

[0079] The Identity by State (IBS) values ​​between two individuals were calculated using PLINK software to obtain the IBS distance matrix. Based on the IBS distance matrix, the phylogenetic tree was constructed using the neighbor-joining method of PHYLIP software, and finally the phylogenetic tree was visualized using Figtree software. The phylogenetic tree results are shown in Figure 1. Figure 4 As shown. The results show that each pig breed can be clearly distinguished and can be used for breed identification. In addition, the three commercial pig breeds (Duroc, Large White and Landrace) are in a large evolutionary branch, while all Chinese local breeds are in another large evolutionary branch. The Beijing Black Pig is located in the middle between these two main evolutionary branches, which is consistent with the breeding history of the Beijing Black Pig. The results show that the chip provided by the present invention can be well applied to the genetic classification and evolutionary analysis of the Beijing Black Pig, and the identification results are accurate and reliable.

[0080] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0081] Industrial Applicability

[0082] The present invention provides a combination of SNP molecular markers for Beijing black pig genotyping, a chip, and a preparation method and application thereof. The combination of SNP molecular markers for Beijing black pig genotyping of the present invention is composed of 49,963 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-49,963, respectively. The molecular marker combination can be used to prepare a Beijing black pig molecular breeding chip, and can be further applied to the specific identification of Beijing black pigs and other pig breeds (European commercial pig breeds, Chinese local pig breeds), target trait QTL, associated sites and candidate gene identification, kinship identification and family division, etc., with good economic value and application prospects.

Claims

1. A combination of SNP molecular markers for Beijing black pig genotyping, characterized in that: It consists of 49,963 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-49,963 respectively.

2. Use of the DNA probe corresponding to the SNP molecular marker combination according to claim 1 in preparing a molecular breeding chip for Beijing black pigs.

3. A 50K SNP chip for molecular breeding of Beijing black pigs, characterized in that: A DNA probe corresponding to the SNP molecular marker combination according to claim 1, wherein the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-49,963 respectively.

4. The SNP molecular marker combination for Beijing black pig genotyping according to claim 1 or the Beijing black pig molecular breeding 50K SNP chip according to claim 3, characterized in that: The SNP site of the SNP molecular marker is located at the 71st position of the nucleotide sequence shown in SEQ ID NO.1-49,963.

5. Use of the SNP molecular marker combination for Beijing black pig genotyping according to claim 1 or the Beijing black pig molecular breeding 50K SNP chip according to claim 3 in Beijing black pig genome-wide association analysis.

6. The use according to claim 5, characterized in that In the whole genome association analysis of Beijing black pig, the Beijing black pig molecular breeding 50KSNP chip described in claim 3 is used to perform genotype detection on Beijing black pig, and the relevant phenotypic traits are determined. The whole genome association analysis is performed based on the linear mixed model. The expression of the linear mixed model is: y=Xα+Yβ+Zγ+e y is the phenotype vector, α is the SNP marker effect vector, X is the association matrix corresponding to α, coded as 0, 1, 2, β is the non-genetic fixed effect vector, Y is the association matrix corresponding to β, and γ is the residual polygenic effect vector, which follows a normal distribution G is the genomic kinship matrix, is the additive genetic variance, Z is the correlation matrix corresponding to γ, e is the residual vector, and obeys I is the identity matrix, are random residuals.

7. Use of the SNP molecular marker combination for Beijing black pig genotyping according to claim 1 or the Beijing black pig molecular breeding 50K SNP chip according to claim 3 in Beijing black pig variety identification.

8. The use according to claim 7, characterized in that When identifying pigs of unknown breed, the Beijing black pig molecular breeding 50K SNP chip described in claim 3 is used to perform genotype detection on the unknown breed of pigs, and the genotype is merged with the chip data of the known Beijing black pig. The chip data is quality controlled and the IBS distance matrix between individuals is calculated using PLINK software. Based on the IBS distance matrix, a phylogenetic tree is constructed using the neighbor-joining method of PHYLIP software. Finally, the phylogenetic tree is visualized using Figtree software to determine whether the unknown breed of pig is a Beijing black pig, thereby performing breed identification.

9. Use of the SNP molecular marker combination for Beijing black pig genotyping according to claim 1 or the Beijing black pig molecular breeding 50K SNP chip according to claim 3 in Beijing black pig breeding.

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