WO2023201950A1 - 一种用于北京黑猪基因分型的snp分子标记组合、芯片及其制备方法与应用 - Google Patents

一种用于北京黑猪基因分型的snp分子标记组合、芯片及其制备方法与应用 Download PDF

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WO2023201950A1
WO2023201950A1 PCT/CN2022/115226 CN2022115226W WO2023201950A1 WO 2023201950 A1 WO2023201950 A1 WO 2023201950A1 CN 2022115226 W CN2022115226 W CN 2022115226W WO 2023201950 A1 WO2023201950 A1 WO 2023201950A1
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snp
beijing black
pig
chip
black pig
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刘剑锋
刘振
周磊
黄千千
冯文
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中国农业大学
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  • the invention relates to the technical fields of genomics, bioinformatics, molecular biology and genome breeding. Specifically, it relates to a SNP molecular marker combination and chip for genotyping Beijing black pigs and their preparation methods and applications.
  • Beijing black pigs are bred by using foreign pig breeds such as Large White pigs, Berkshire pigs, Soviet Large White pigs and Caucasian pigs, and crossing them with local pig breeds to produce a large number of offspring. This breed was officially named "Beijing Black Pig” in 1982, and was later designated as the original maternal line pig.
  • Beijing Black Pig combines the excellent traits of domestic and foreign pig breeds, combining the pig breed's characteristics of tolerance to rough feeding, early maturity and prolific yield, and tender meat, with the characteristics of large body, fast growth, and meatiness.
  • SNP refers to the variation of a single nucleotide at the genome level, including molecular markers formed by single base deletions, insertions, conversions, and transversions. It is characterized by a large number and wide distribution. As genetic markers, SNPs contribute to the genetic variation of complex traits and are therefore widely used in genetic research. SNP chips are made by fixing fluorescently labeled DNA probes on silicon wafers, and then perform SNP typing by hybridizing the probe DNA with genomic DNA. SNPs bind to probes on the surface of the silicon wafer rather than the genome sequence, allowing the DNA of a large number of individuals to be immobilized onto a single chip for analysis.
  • Illumina's Infinium SNP chip technology is currently a relatively mature and widely used whole-gene SNP detection platform.
  • the denatured DNA is hybridized with the probe on the chip beads, then the unhybridized DNA is removed, and the obtained DNA is subjected to an extension reaction of specially labeled bases; finally, the SNP is combined with the immunological binding of the label and the fluorescent group. The information is converted into visible fluorescence information.
  • the mature commercial SNP chips in pigs are mainly based on the PorcineSNP60 chip developed by geneseek company on the Illumina platform, the GGP-Porcine HD chip, and the compass porcineSNP55 chip designed by Compson.
  • Another platform is the Affymetrix platform.
  • pigs mainly use high-density chips developed by Affymetrix, which contain about 650,000 SNPs. They are expensive and rarely used.
  • the purpose of the present invention is to provide a SNP molecular marker combination, chip, and preparation method and application for genotyping Beijing black pigs.
  • the present invention provides a SNP molecular marker combination for Beijing black pig genotyping, which consists of 49,963 SNP molecular markers.
  • the nucleotide sequences of the SNP molecular markers are as follows: SEQ ID NO: 1- 49,963 shown.
  • the present invention provides the application of the above-mentioned SNP molecular marker combination in preparing a Beijing black pig molecular breeding chip.
  • the present invention provides a Beijing black pig molecular breeding 50K SNP chip, which contains 1-49,963 SNP molecular markers.
  • the nucleotide sequences of the SNP molecular markers are as shown in SEQ ID NO: 1-49,963.
  • the 71st base of each sequence is the 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 on the SMIC One PLUS pig breeding chip, including 23,299 SNP sites; the second category, derived from sites reported in existing literature and public databases that are significantly related to existing important economic traits (especially meat quality traits) (exons and regulatory regions are preferred), including 937 SNPs Sites; the third category, derived from the SNP variation detection results of resequencing data of Beijing black pigs and commercial pig breeds, sites with large differences in absolute allele frequencies, including 2,536 SNP sites; the fourth category , derived from the Beijing Black Pig genome-wide association analysis results, loci significantly related to meat quality traits, including 31 SNP sites; the fifth category, derived from the Beijing Black Pig resequencing data for SNP variation detection
  • the present invention provides a method for preparing the above-mentioned Beijing black pig molecular breeding 50K SNP chip, which includes the following steps:
  • Step 1 Obtain the first type of probe: use SMIC One PLUS pig breeding chip (available from Beijing Compson Biotechnology Co., Ltd.) detects the Beijing black pig population and performs quality control on the chip data.
  • the quality control standards are: eliminate SNP detection rate ⁇ 95%, minimum, etc. SNP sites with gene frequency ⁇ 0.1, Hardy-Weinberg equilibrium test P value ⁇ 10 -6 , and SNP sites without chromosome position information; SMIC-1 PLUS pig breeding chip position information has been changed from Sus scrofa 10.2 version to Sus scrofa 11.1 version Refer to the genome to obtain the first type of candidate SNP;
  • Step 2 Obtaining the second type of probe: Combining the public database NCBI and QTL database 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 are significant SNP, and based on Beijing black pig resequencing data, sites with MAF ⁇ 0.05 were eliminated to obtain the second type of candidate SNP;
  • Step 3 Obtaining the third type of probe: Based on the resequencing data of Beijing black pigs, large white pigs and landrace pigs, screen the sites where the absolute allele frequency difference between Beijing black pigs, large white pigs and landrace pigs is greater than or equal to 0.6 , and based on the Beijing black pig resequencing data, sites with MAF ⁇ 0.05 were eliminated to obtain the third type of candidate SNP;
  • Step 4 Obtain the fourth type of probe: Use the SMIC One PLUS pig breeding chip to detect the Beijing black pig population, and conduct a genome-wide association analysis based on the Beijing black pig chip data and meat quality phenotype data.
  • the meat quality phenotype data includes muscle Internal fat, protein content, moisture content, collagen content, dripping loss, meat color and pH value 24 hours after slaughter (a total of 7 traits); loci significantly related to meat quality traits identified by genome-wide association analysis, as the third Four categories of alternative SNPs;
  • Step 5 Obtaining the fifth category of probes: Based on Beijing black pig resequencing data, retain SNPs with MAF ⁇ 0.1 to obtain the fifth category of candidate SNPs;
  • Step 6 In order to make the SNP sites evenly distributed at the whole genome level, divide the autosome and X chromosome into a 48kb window, and select 1 SNP to fill each window according to the priority of the first to fifth types of probes. ;
  • Step 7 Score the screened SNP sites by the Infmium iSelect scoring system (http://www.ill umina.com/), and remove sites with intra-gene scores ⁇ 0.7 and inter-gene scores ⁇ 0.9; for After deleting the unqualified SNP sites, select the closest SNP sites to supplement them, and score them again until all sites are qualified. The obtained tag sequences will then be used for the production of SNP chips.
  • Infmium chip manufacturing technology is used to produce SNP chips. Specifically, the obtained tag sequences can be handed over to Illumina to design and produce Infmium SNP chips.
  • the present invention provides the above-mentioned SNP molecular marker combination for Beijing black pig genotyping or the application of the Beijing black pig molecular breeding 50K SNP chip in the whole genome association analysis of Beijing black pig.
  • the present invention uses the above-mentioned Beijing black pig molecular breeding 50K SNP chip to detect the genotype of Beijing black pigs, and determines the relevant phenotypic traits, and conducts whole genome association analysis based on the linear mixed model.
  • the expression of the mixed model is:
  • y is the phenotype vector
  • ⁇ is the SNP marker effect vector
  • X is the correlation matrix corresponding to ⁇ , coded as 0, 1, 2
  • ⁇ is the non-genetic fixed effect vector
  • Y is the correlation matrix corresponding to ⁇
  • ⁇ is the residual Polygenic effect vector, obeying normal distribution
  • G is the genomic kinship matrix
  • Z is the correlation matrix corresponding to ⁇
  • e is the residual vector, obeying I is the identity matrix, is the random residual.
  • the present invention provides the above-mentioned SNP molecular marker combination for Beijing black pig genotyping or the application of the Beijing black pig molecular breeding 50K SNP chip in the identification of Beijing black pig varieties.
  • the present invention uses the above-mentioned Beijing Black Pig Molecular Breeding 50K SNP chip to perform genotype detection on the pigs of unknown breeds, and merges them with the chip data known to be Beijing Black Pigs, using PLINK
  • the software controls the quality of the chip data and calculates the IBS distance matrix between individuals. Based on the IBS distance matrix, the neighbor-joining method of PHYLIP software is used to construct a phylogenetic tree. Finally, the phylogenetic tree is visualized through Figtree software to determine whether the unknown breed of pig is Beijing black pig for breed identification.
  • Quality control standards are 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 application of the chip of the present invention can realize the breed identification of Beijing black pig and other pig breeds (European commercial pig breeds, Chinese local pig breeds).
  • the present invention provides the above-mentioned SNP molecular marker combination for Beijing black pig genotyping or the application of the Beijing black pig molecular breeding 50K SNP chip in Beijing black pig breeding.
  • the specialized SNP chip for Beijing black pig molecular breeding of the present invention is designed based on its own needs.
  • the source of SNP sites is more in line with the population characteristics, and the chip density is reduced by selecting the effective number of SNPs, thereby saving costs to the greatest extent.
  • the detection cost of the chip of the present invention is 150 yuan/piece, which is 50 yuan/piece lower than the cost of SMIC One PLUS pig breeding chip.
  • the chip site made with the SNP molecular marker combination of the present invention is more evenly distributed: the position information of SMIC One PLUS pig breeding chip is changed from Sus scrofa 10.2 version is converted to Sus scrofa 11.1 version reference genome, and the standard deviation of the distance between adjacent SNPs is 50.4kb, indicating that the degree of discreteness of the distance between adjacent SNPs is large; while the standard deviation of the distance between adjacent SNPs in the chip of the present invention is 9.4kb, It is much lower than the SMIC One PLUS pig breeding chip, indicating that the distance between adjacent SNPs is less discrete and distributed more evenly throughout the genome.
  • the SNP site of the chip of the present invention has better polymorphism in the Beijing black pig population.
  • the minimum allele frequency of the SMIC-1 PLUS pig breeding chip in the Beijing black pig population is 0.220 on average.
  • the chip of the present invention is 0.276.
  • the SNP molecular marker combination provided by the invention has the characteristics of uniform distribution of SNP sites and high whole-genome coverage. Using these SNP markers for whole-genome breeding can effectively improve the degree of correlation with traits through linkage disequilibrium, ensuring the breeding value. Accuracy of estimate.
  • the present invention screens sites based on resequencing data of Beijing black pigs. 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), improving the practicability of the chip.
  • the present invention uses resequencing data of Beijing black pigs and commercial pig breeds to screen out sites with large absolute allele frequency differences between breeds, which can be used for breed identification of Beijing black pigs and is beneficial to the protection of Beijing black pigs.
  • Germplasm resources can also be used for whole-genome selective breeding of Beijing black pigs, identification of QTL, associated loci and candidate genes for target traits of Beijing black pigs, genetic relationship identification, family classification, etc., and has broad application prospects.
  • Figure 1 is a histogram of the minimum allele frequency (MAF) frequency (Frequency) distribution of SNP sites in Example 1 of the present invention.
  • MAF minimum allele frequency
  • Figure 2 is a box plot (unit: kb) of the distance between adjacent SNP sites in Example 1 of the present invention.
  • the ordinate is the SNP interval (Interval) and the abscissa is the chromosome (CHR) number.
  • Figure 3 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, and -log10 transformation is performed.
  • the abscissa is the chromosome number.
  • Figure 4 is a phylogenetic tree diagram of Beijing black pig, European commercial pig species and Chinese local pig species in Example 3 of the present invention.
  • the abbreviation represents the breed information, and the numerical number is the individual number 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 stands for Blue Tang pig
  • LC stands for Luchuan pig
  • MIN stands for Min pig.
  • SNP site screening process In order to make SNP sites evenly distributed at the whole genome level, the autosomes and X chromosomes are divided into 48kb windows. Each window is selected according to the priority of the first to fifth type probes. SNP filling.
  • the quality control standards are: elimination of SNP detection rate ⁇ 95%, minimum allele frequency ⁇ 0.1, Hardy-Weinberg equilibrium SNP sites with P values ⁇ 10 -6 and no chromosomal location information were tested.
  • the location information of SMIC One PLUS pig breeding chip is Sus scrofa 10.2 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000003025.5/) version reference genome, which can be obtained through the UCSC online website (http://genome .ucsc.edu/cgi-bin/hgLiftOver) to convert the position information to the Sus scrofa 11.1 version (https://www.ncbi.nlm.nih.gov/assembly/GCF_000003025.6/) reference genome to obtain the first type of preparation Choose SNP.
  • the length of the whole pig genome (excluding Y chromosome) is about 2.4Gb.
  • the present invention divides the whole length of the pig genome into a window every 48kb.
  • the specific method for determining SNP 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, then select The SNP directly becomes the final target SNP; 3) If there is no candidate SNP for the first type of probe in the window, the window is retained and waits for the candidate SNP for the second type of probe to be filled. 4) Set the filtering standard of 20k ⁇ the distance between adjacent SNPs/its window interval ⁇ 75k, and SNPs that do not meet this standard will be eliminated. A final total of 23,299 SNPs were identified for category I probes.
  • the window After completing the screening of the first type of probes, populate the window with no SNPs with 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 one closest to the midpoint of the window. SNP; 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 for the second type of probe in the window, the window is retained and waits Alternative SNP population for type III probes. 4) Set the filtering standard of 20k ⁇ the distance between adjacent SNPs/its window interval ⁇ 75k, and SNPs that do not meet this standard will be eliminated. Finally, 937 SNPs of type II probes were identified.
  • the specific method is as follows: 1) If there are more than or equal to 2 candidate SNPs in the window, select the one closest to the midpoint of the window. SNP; 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 for the third type of probe in the window, the window is retained and waits Alternative SNP population for category IV probes. 4) Set the filtering standard of 20k ⁇ the distance between adjacent SNPs/its window interval ⁇ 75k, and SNPs that do not meet this standard will be eliminated. Finally, 2,536 SNPs of category III probes were identified.
  • Meat quality phenotypic data includes 7 traits including intramuscular fat, protein content, moisture content, collagen content, drip loss, meat color, and pH 24 hours after slaughter. All loci identified by genome-wide association analysis that were significantly related to meat quality traits were retained as the fourth type of probes. Finally, 31 SNPs of category IV probes were determined.
  • 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 window has only 1 SNP, then the SNP directly becomes the target SNP. 3) If there are no candidate SNPs for the fifth category of probes in this window, relax the criteria and use SNPs with MAF ⁇ 0.05 for filling.
  • SNPs with MAF ⁇ 0.05 in the non-homologous regions of the Y chromosome were selected on the basis of considering uniformity, and a total of 15 SNPs were selected. These SNPs are only used for sexing Beijing black pigs when the phenotypic sex information is unknown.
  • This 50K SNP chip has three advantages: First, it has functional correlation with economic traits. The chip collected and identified a large number of significant sites related to pork quality traits, growth traits, reproductive traits, and health traits, which increased the practicality of the chip. Second, the screening sites were based on Beijing black pig resequencing data to ensure that the selected sites were selected. The sites have high polymorphism (the MAF distribution of SNP sites is shown in Figure 1), and the sites are evenly distributed (the distance between adjacent SNP sites is shown in Figure 2), ensuring the accuracy of breeding value estimation; thirdly, It is specific. The chip site is specially designed for the Beijing black pig breed. The sites that are significantly different between Beijing black pig and commercial pig breeds are selected, which can specifically identify Beijing black pig and other pig breeds.
  • the quality control standards are 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 ; SNP site information obtained after quality control will be used for subsequent genome-wide association analysis.
  • the quartile test method was used to eliminate outliers for intramuscular fat traits and carcass weight phenotypic traits.
  • y is the intramuscular fat phenotype vector
  • ⁇ is the SNP marker effect vector
  • Y is the correlation matrix corresponding to ⁇
  • ⁇ is the residual polygenic effect vector, obeying the normal distribution
  • G is the genomic kinship matrix
  • Z is the correlation matrix corresponding to ⁇
  • e is the residual vector, obeying I is the identity matrix, is the random residual.
  • the significance test was performed using the Wald test, with p ⁇ 1 ⁇ 10 -5 as the threshold for the potential genome-wide significance level.
  • the resulting Manhattan results are shown in 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.
  • Example 1 of the present invention 22 Beijing Black Pigs (BJB), 22 Duroc Pigs (DU), 16 Large White Pigs (LW), and 5 Landrace Pigs (LD) were , 5 Mashen pigs (MS), 5 Huainan pigs (HN), 5 Langtang pigs (LT), 5 Luchuan pigs (LC) and 5 Min pigs (MIN), a total of 90 pigs of 9 breeds were extracted. DNA genotype testing.
  • the quality control standards are 1 individual genotype frequency >0.95; 2 SNP call rate (SNP call rate) >0.95; 3 minimum allele frequency (MAF) >0.05; 4 Hardy-Wen The P value of Berg's equilibrium test is >10 -6 ; the SNP site information obtained after quality control is used for subsequent variety identification analysis.
  • the invention provides a SNP molecular marker combination and chip for genotyping Beijing black pigs and their preparation methods and applications.
  • the SNP molecular marker combination used for Beijing black pig genotyping of the present invention consists of 49,963 SNP molecular markers.
  • 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 used to specifically identify Beijing black pigs and other pig breeds (European commercial pig breeds, Chinese local pig breeds), target trait QTL, associated sites and candidates It has good economic value and application prospects in terms of genetic identification, genetic relationship identification and family classification.

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Abstract

涉及分子生物学、功能基因组学、生物信息学、基因组育种技术领域，具体公开了一种用于北京黑猪基因分型的SNP分子标记组合、芯片及其制备方法与应用。涉及用于北京黑猪基因分型的SNP分子标记组合，由49,963个SNP分子标记组成，所述SNP分子标记的核苷酸序列分别如SEQ ID NO：1-49,963所示。应用该分子标记组合可制备北京黑猪分子育种芯片，并可进一步应用于特异性鉴定北京黑猪与其他猪种(欧洲商业猪种、中国地方猪种)、目标性状QTL、关联位点及候选基因鉴定、亲缘关系鉴定和家系划分等方面。

Description

一种用于北京黑猪基因分型的SNP分子标记组合、芯片及其制备方法与应用
交叉引用
本申请要求2022年4月22日提交的专利名称为“一种用于北京黑猪基因分型的SNP分子标记组合、芯片及其制备方法与应用”的第202210433080.4号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及基因组学、生物信息学、分子生物学和基因组育种技术领域，具体地说，涉及一种用于北京黑猪基因分型的SNP分子标记组合、芯片及其制备方法与应用。
背景技术
北京黑猪是利用大白猪、巴克夏猪、苏联大白猪与高加索猪等国外猪种，与本地猪种杂交产生大量后代的基础上培育而成。该品种于1982年正式命名为“北京黑猪”，之后还被定为母系原种猪。北京黑猪融合了国内外猪种的优良种性，将猪种的耐粗饲、早熟多产、肉质香嫩的特点，和体大快长、多肉的特性结合起来。
然而，当前北京黑猪核心群群体规模过小，难以实施有效的育种选育，群体生产性能亟需进一步提高。开发一款北京黑猪分子育种专门化芯片，实现北京黑猪特色品种的合理开发利用，培育专门化北京黑猪优良品种及优质猪配套系，发展北京黑猪的种质资源，并基于该品种开发优质高档的猪肉产品，对于加速北京黑猪的选育改良，促进特色畜牧业发展有重大意义。
SNP是指基因组水平上单个核苷酸的变异，包括单碱基的缺失、插入、转换及颠换等形成的分子标记，具有数量大分布广的特点。SNP作为遗传标记，对复杂性状的遗传变异具有贡献，因此被广泛应用于遗传研究中。SNP芯片是将带有荧光标记的DNA探针固定在硅片上制作而成，再通过探针DNA与基因组DNA杂交进行SNP分型。SNP与硅片表面的探针结合而不是基因组序列，这样可以将大量个体的DNA固定到一张芯片上分析。
Illumina公司的Infinium SNP芯片技术是目前比较成熟和应用广泛的全基因 SNP检测平台。首先将变性的DNA与芯片微珠上的探针进行杂交，然后去除未杂交结合的DNA，并将获得的DNA进行特殊标记碱基的延伸反应；最后通过标记和荧光基团的免疫结合将SNP信息转化为可视的荧光信息。
目前，猪上成熟的商用SNP芯片主要基于illumina平台的geneseek公司开发的PorcineSNP60芯片、GGP-Porcine HD芯片、康普森公司设计的compass porcineSNP55芯片。另一个平台是Affymetrix平台，目前猪上主要有Affymetrix开发的高密度芯片，含大约650,000个SNP，价格高昂，使用量很少。
然而，当前的商业化芯片由于SNP位点来源具有一定的局限性，且大多针对于大白猪、长白猪、杜洛克等国外猪种设计，对于北京黑猪这一品种适应性较差。因此，有必要针对性地对适用于北京黑猪的芯片进行研发。
发明内容
本发明的目的是提供一种北京黑猪基因分型的SNP分子标记组合、芯片及其制备方法与应用。
具体地，本发明的技术方案如下：
第一方面，本发明提供一种用于北京黑猪基因分型的SNP分子标记组合，其由49,963个SNP分子标记组成，所述SNP分子标记的核苷酸序列分别如SEQ ID NO：1-49,963所示。
第二方面，本发明提供上述SNP分子标记组合在制备北京黑猪分子育种芯片中的应用。
第三方面，本发明提供一种北京黑猪分子育种50K SNP芯片，其包含1-49,963个SNP分子标记，所述SNP分子标记的核苷酸序列分别如SEQ ID NO：1-49,963所示。
上述用于北京黑猪基因分型的SNP分子标记组合或北京黑猪分子育种50K SNP芯片，其中，所述SNP分子标记的SNP位点位于SEQ ID NO.1-49,963所示的核苷酸序列的第71位。
本发明提供的北京黑猪分子育种50K SNP芯片，其中，每条序列第71位碱基为SNP位点。所述SNP分子标记主要来自于五类SNP位点：第一类，来源于中芯一号PLUS猪育种芯片在北京黑猪群体中多态性良好和检出率高的位点，包 含23,299个SNP位点；第二类，来源于已有文献和公开数据库报道的与现有重要经济性状(特别是肉质性状)显著相关的位点(外显子区和调控区优先)，包含937个SNP位点；第三类，来源于由北京黑猪与商业猪种重测序数据进行SNP变异检测的结果中，绝对等位基因频率差异较大的位点，包含2,536个SNP位点；第四类，来源于由北京黑猪全基因组关联分析结果中，与肉质性状显著相关的位点，包含31个SNP位点；第五类，来源于由北京黑猪重测序数据进行SNP变异检测的位点，包含23,160个SNP位点。以上5类探针共计包含49,963个SNP位点。
第四方面，本发明提供一种制备上述北京黑猪分子育种50K SNP芯片的方法，其包括以下步骤：
步骤①：第一类探针的获得：使用
Figure PCTCN2022115226-appb-000001
中芯一号PLUS猪育种芯片(可购自北京康普森生物技术有限公司)检测北京黑猪群体，对芯片数据进行质控，质控标准为:剔除SNP检出率＜95％、最小等位基因频率＜0.1、哈代-温伯格平衡检验P值＜10 -6以及没有染色体位置信息的SNP位点；中芯一号PLUS猪育种芯片位置信息从Sus scrofa 10.2版本转为Sus scrofa 11.1版本参考基因组，得到第一类备选SNP；
步骤②：第二类探针的获得：结合公共数据库NCBI和QTLdatabase获得与猪肉质性状、生长性状、繁殖性状及健康性状相关候选基因内部及上下游SNP位点、全基因组关联分析和QTL结果显著SNP，并基于北京黑猪重测序数据，剔除MAF<0.05的位点，得到第二类备选SNP；
步骤③：第三类探针的获得：基于北京黑猪、大白猪与长白猪的重测序数据，筛选北京黑猪与大白猪、长白猪的绝对等位基因频率差异均大于等于0.6的位点，并基于北京黑猪重测序数据，剔除MAF<0.05的位点，得到第三类备选SNP；
步骤④：第四类探针的获得：使用中芯一号PLUS猪育种芯片检测北京黑猪群体，基于北京黑猪的芯片数据和肉质表型数据进行全基因组关联分析，肉质表型数据包括肌内脂肪、蛋白含量、水分含量、胶原蛋白含量、滴水损失、肉色和屠宰后24小时pH值(共7个性状)；由全基因组关联分析鉴定到的与肉质性状显著相关的位点，作为第四类备选SNP；
步骤⑤：第五类探针的获得：基于北京黑猪重测序数据，保留MAF≥0.1 的SNP，得到第五类备选SNP；
步骤⑥：为了使SNP位点在全基因组水平上均匀分布，将常染色体与X染色体分为48kb一个窗口，每个窗口按照第一类至第五类探针的优先级，选择1个SNP填充；
步骤⑦：将筛选好的SNP位点由Infmium iSelect打分系统(http://www.ill umina.com/)打分，将基因内分值<0.7和基因间分值<0.9的位点去掉；对于删掉的不合格的SNP位点，选取距离其最近的SNP位点进行补充，并且再次进行打分，直至全部位点合格，之后将获得的标签序列用于SNP芯片的制作。
本发明中利用Infmium芯片制造技术制作SNP芯片，具体可将获得的标签序列交由Illumina公司设计制作Infmium SNP芯片。
第五方面，本发明提供上述用于北京黑猪基因分型的SNP分子标记组合或北京黑猪分子育种50K SNP芯片在北京黑猪全基因组关联分析中的应用。
本发明在进行北京黑猪全基因组关联分析中，应用上述北京黑猪分子育种50K SNP芯片对北京黑猪进行基因型检测，并测定相关表型性状，基于线性混合模型进行全基因组关联分析，线性混合模型的表达式为：
y＝Xα+Yβ+Zγ+e
y是表型向量，α为SNP标记效应向量，X是对应于α的关联矩阵，编码为0、1、2，β为非遗传固定效应向量，Y是对应于β的关联矩阵，γ是剩余多基因效应向量，服从正态分布
Figure PCTCN2022115226-appb-000002
G是基因组亲缘关系矩阵，
Figure PCTCN2022115226-appb-000003
是加性遗传方差，Z是对应于γ的关联矩阵，e为残差向量，服从
Figure PCTCN2022115226-appb-000004
I为单位矩阵，
Figure PCTCN2022115226-appb-000005
是随机残差。
第六方面，本发明提供上述用于北京黑猪基因分型的SNP分子标记组合或北京黑猪分子育种50K SNP芯片在北京黑猪品种鉴定中的应用。
本发明在进行未知品种的猪的鉴定时，应用上述北京黑猪分子育种50K SNP芯片对所述未知品种的猪进行基因型检测，与已知确定为北京黑猪的芯片数据进行合并，使用PLINK软件对芯片数据质控和计算个体间的IBS距离矩阵，并基于IBS距离矩阵，使用PHYLIP软件的邻接法构建系统发育树，最后通过Figtree软件可视化系统发育树，判断所述未知品种的猪是否为北京黑猪，从而进行品种鉴定。
质控标准为①个体基因型频率>0.95；②SNP检出率(SNP call rate)>0.95；③最小等位基因频率(MAF)>0.05；④哈代-温伯格平衡检验P值>10 -6。
应用本发明芯片可实现北京黑猪与其他猪种(欧洲商业猪种、中国地方猪种)品种鉴定。
第七方面，本发明提供上述用于北京黑猪基因分型的SNP分子标记组合或北京黑猪分子育种50K SNP芯片在北京黑猪育种中的应用。
本发明的有益效果至少在于：
本发明的北京黑猪分子育种专门化SNP芯片结合自身需要进行设计，SNP位点来源更加符合群体特征，且通过选择有效的SNP数目降低了芯片密度，从而最大程度地节约成本。本发明芯片检测费用为150元/张，成本较中芯一号PLUS猪育种芯片降低了50元/张。
此外，与当前猪主流50k SNP芯片——中芯一号PLUS猪育种芯片相比，以本发明SNP分子标记组合制作的芯片位点分布更均匀：将中芯一号PLUS猪育种芯片位置信息从Sus scrofa 10.2版本转为Sus scrofa 11.1版本参考基因组，相邻SNP距离的标准差为50.4kb，说明相邻SNP距离的离散程度较大；而本发明芯片相邻SNP距离的标准差为9.4kb，远低于中芯一号PLUS猪育种芯片，说明相邻SNP距离的离散程度更小，在全基因组分布更均匀。
从多态性角度，本发明芯片的SNP位点在北京黑猪群体中的多态性更好，中芯一号PLUS猪育种芯片在北京黑猪群体中的最小等位基因频率平均为0.220，而本发明芯片为0.276。
本发明提供的SNP分子标记组合具有SNP位点分布均匀，全基因组覆盖率高的特点，利用这些SNP标记进行全基因组育种，能有效地通过连锁不平衡提高与性状的关联程度，保证了育种值估计的准确性。本发明筛选位点时基于北京黑猪的重测序数据，所选位点在北京黑猪群体中有较高的多态性，保证了芯片位点的高质量。本款芯片增加了与猪经济性状(特别是肉质性状)相关的显著SNP位点，提高了芯片的实用性。本发明筛选SNP位点时使用北京黑猪和商业猪种的重测序数据，筛选出品种间绝对等位基因频率差异大的位点，可用于北京黑猪的品种鉴定，有利于保护北京黑猪种质资源。此外，该款芯片还可用于北京黑猪全基因组选择育种、北京黑猪目标性状QTL、关联位点及候选基因鉴定，亲缘 关系鉴定、家系划分等方面，具有广阔的应用前景。
附图说明
图1为本发明实施例1中SNP位点的最小等位基因频率(MAF)频数(Frequency)分布直方图。
图2为本发明实施例1中相邻SNP位点距离的箱线图(单位：kb)，纵坐标为SNP间隔(Interval)，横坐标为染色体(CHR)编号。
图3为本发明实施例2中北京黑猪肌内脂肪性状全基因组关联分析结果的曼哈顿图，纵坐标为各SNP位点的p值，并进行-log10转换，横坐标为染色体编号。
图4为本发明实施例3中北京黑猪与欧洲商业猪种、中国地方猪种系统发育树图。其中字母缩写代表品种信息，数字编号为每个品种的个体编号，BJB代表北京黑猪，DU代表杜洛克猪，LW代表大白猪，LD代表长白猪，MS代表马身猪，HN代表淮南猪，LT代表蓝塘猪，LC代表陆川猪，MIN代表民猪。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。若未特别指明，各实施例中所用的设备和试剂均常规市售可得。
实施例1北京黑猪分子育种50K SNP芯片的制备方法
SNP位点筛选流程：为了使SNP位点在全基因组水平上均匀分布，将常染色体与X染色体分为48kb一个窗口，每个窗口按照第一类至第五类探针的优先级，选择1个SNP填充。
1、第一类探针的获得：
使用中芯一号PLUS猪育种芯片检测北京黑猪群体，对芯片数据进行质控，质控标准为:剔除SNP检出率＜95％、最小等位基因频率＜0.1、哈代-温伯格平 衡检验P值＜10 -6以及没有染色体位置信息的SNP位点。
中芯一号PLUS猪育种芯片位置信息为Sus scrofa 10.2(https://www.ncbi.nlm.nih.gov/assembly/GCF_000003025.5/)版本参考基因组，通过UCSC在线网站(http://genome.ucsc.edu/cgi-bin/hgLiftOver)将位置信息转为Sus scrofa 11.1版本(https://www.ncbi.nlm.nih.gov/assembly/GCF_000003025.6/)参考基因组，得到第一类备选SNP。
猪的全基因组(不含Y染色体)长度约为2.4Gb，为了保证这些SNP在全基因组水平上尽可能均匀分布，本发明将猪全基因组全长以每48kb划分为一个窗口。具体确定SNP的方法：1)若该窗口里有大于等于2个的备选SNP，选取距离该窗口中点最近的SNP；2)若该窗口里的备选SNP数为1个，则备选SNP直接成为最终目标SNP；3)若该窗口无第一类探针的备选SNP，则保留该窗口，等待第二类探针的备选SNP填充。4)设置20k≤相邻SNP的间距/其窗口间隔≤75k这一过滤标准，不符合该标准的SNP进行剔除。最终确定了23,299个第一类探针的SNP。
2、第二类探针的获得：
结合公共数据库NCBI(https://www.ncbi.nlm.nih.gov/pubmed/)、QTLdatabase(https://www.animalgenome.org/cgi-bin/QTLdb/index/)获得与猪肉质性状、生长性状、繁殖性状及健康形状等经济性状相关候选基因内部及上下游SNP位点(外显子区和调控区优先)、全基因组关联分析和QTL结果显著SNP。并基于北京黑猪重测序数据，剔除MAF<0.05的位点，得到第二类备选SNP。
完成第一类探针筛选后，对于尚无SNP的窗口，填充第二类探针，具体方法如下：1)若该窗口里有大于等于2个的备选SNP，选取距离该窗口中点最近的SNP；2)若该窗口里的备选SNP数为1个，则备选SNP直接成为最终目标SNP；3)若该窗口无第二类探针的备选SNP，则保留该窗口，等待第三类探针的备选SNP填充。4)设置20k≤相邻SNP的间距/其窗口间隔≤75k这一过滤标准，不符合该标准的SNP进行剔除。最终确定了937个第二类探针的SNP。
3、第三类探针的获得：
基于北京黑猪、大白猪与长白猪的重测序数据，筛选北京黑猪与大白猪、长白猪的绝对等位基因频率差异均大于等于0.6的位点。并基于北京黑猪重测序数 据，剔除MAF<0.05的位点，得到第三类备选SNP。
完成第二类探针筛选后，对于尚无SNP的窗口，填充第三类探针，具体方法如下：1)若该窗口里有大于等于2个的备选SNP，选取距离该窗口中点最近的SNP；2)若该窗口里的备选SNP数为1个，则备选SNP直接成为最终目标SNP；3)若该窗口无第三类探针的备选SNP，则保留该窗口，等待第四类探针的备选SNP填充。4)设置20k≤相邻SNP的间距/其窗口间隔≤75k这一过滤标准，不符合该标准的SNP进行剔除。最终确定了2,536个第三类探针的SNP。
4、第四类探针的获得：
基于北京黑猪的芯片数据和肉质表型数据进行全基因组关联分析。肉质表型数据包括肌内脂肪、蛋白含量、水分含量、胶原蛋白含量、滴水损失、肉色、屠宰后24小时pH共7个性状。全基因组关联分析鉴定到的与肉质性状显著相关的位点，全部保留作为第四类探针。最终确定了31个第四类探针的SNP。
5、第五类探针的获得：
基于北京黑猪重测序数据，保留MAF≥0.1的SNP，得到第五类备选SNP。
对于尚无SNP的窗口，填充第五类探针，具体方法如下：1)若该窗口里有大于等于2个的备选SNP，选取距离该窗口中点最近的SNP；2)若该窗口只有1个SNP，则该SNP直接成为目标SNP。3)若该窗口无第五类探针的备选SNP，则放宽标准，使用MAF≥0.05的SNP进行填充。
最后，基于北京黑猪重测序数据，在考虑均匀的基础上，挑选位于Y染色体非同源区域MAF≥0.05的SNP，共计挑选了15个SNP。这些SNP仅用于当表型的性别信息未知时，对北京黑猪进行性别鉴定。
综上，最终确定23,160个第五类探针的SNP。
将筛选好的SNP位点交给Illumina公司由Infmium iSelect打分系统(http://www.illumina.com/)打分，将基因内分值<0.7和基因间<0.9的位点去掉。对于删掉的不合格的SNP位点，选取距离其最近的SNP位点进行补充，并且再次进行打分。按照以上步骤鉴定和筛选，最后共获得49,963个SNP位点。按照Illumina Infmium iSelect HD设计要求需要49,963种微珠(beads)。所有49,963个SNP位点为SEQ ID NO:1-49,963所示的49,963条DNA序列。将这些标签序列交由Illumina公司设计制作Infmium SNP芯片。芯片位点在各染色体分布数量见 表1。
该款50K SNP芯片具有三个优点：一是具有经济性状的功能相关性。芯片搜集并确定了大量与猪肉质性状、生长性状、繁殖性状及健康性状等相关的显著位点，增加了芯片的实用性；二是筛选位点时基于北京黑猪重测序数据，保证所选位点有较高的多态性(SNP位点的MAF分布见图1)，且位点分布均匀(相邻SNP位点的距离见图2)，保证了育种值估计的准确性；三是具有特异性，芯片位点专门针对于北京黑猪这一品种进行设计，选取了北京黑猪与商业猪种有较大差异的位点，可以特异性鉴定北京黑猪与其他猪种。
表1芯片位点在各染色体分布数量
染色体 SNP位点数量 染色体 SNP位点数量 染色体 SNP位点数量
1 5,736 8 2,906 15 2,936
2 3,176 9 2,917 16 1,671
3 2,777 10 1,448 17 1,328
4 2,731 11 1,654 18 1,170
5 2,184 12 1,291 X 2,595
6 3,569 13 4,352 Y 15
7 2,543 14 2,964 总计 49,963
实施例2北京黑猪分子育种50K SNP芯片在全基因组关联分析中的应用
使用本发明实施例1提供的北京黑猪分子育种50K SNP芯片对北京黑猪做基因型检测，获取基因型数据。对北京黑猪肌内脂肪性状和北京黑猪的胴体重性状进行测定，获取表型数据。
利用Beagle软件对芯片数据进行自身填充，再利用PLINK软件对其进行质量控制，质控标准为①个体基因型频率>0.95；②SNP检出率(SNP call rate)>0.95；③最小等位基因频率(MAF)>0.05；④哈代-温伯格平衡检验P值>10 -6；质控后得到的SNP位点信息，用于后续全基因组关联分析。
使用四分位检验法剔除肌内脂肪性状和胴体重表型性状的异常值。
使用GEMMA软件基于线性混合模型进行全基因组关联分析。线性混合模型的表达式为：
y＝Xα+Yβ+Zγ+e
y是肌内脂肪表型向量，α为SNP标记效应向量，X是对应于α的关联矩阵，编码为0、1、2，β为非遗传固定效应向量，包含群体均值、性别、胴体重，Y 是对应于β的关联矩阵，γ是剩余多基因效应向量，服从正态分布
Figure PCTCN2022115226-appb-000006
G是基因组亲缘关系矩阵，
Figure PCTCN2022115226-appb-000007
是加性遗传方差，Z是对应于γ的关联矩阵，e为残差向量，服从
Figure PCTCN2022115226-appb-000008
I为单位矩阵，
Figure PCTCN2022115226-appb-000009
是随机残差。
显著性检验采用Wald检验，以p<1×10 -5作为潜在全基因组显著水平的阈值，得到的曼哈顿结果如图3所示。结果显示，位于1号染色体上的全基因组显著关联位点可能是肌内脂肪这一性状的调控位点。
实施例3北京黑猪分子育种50K SNP芯片进行品种鉴定
使用本发明实施例1提供的北京黑猪分子育种50K SNP芯片对22头北京黑猪(BJB)、22头杜洛克猪(DU)、16头大白猪(LW)、5头长白猪(LD)、5头马身猪(MS)、5头淮南猪(HN)、5头蓝塘猪(LT)、5头陆川猪(LC)和5头民猪(MIN)共计9个品种90头猪提取的DNA进行基因型检测。
使用PLINK软件对芯片数据进行质量控制，质控标准为①个体基因型频率>0.95；②SNP检出率(SNP call rate)>0.95；③最小等位基因频率(MAF)>0.05；④哈代-温伯格平衡检验P值>10 -6；质控后得到的SNP位点信息，用于后续品种鉴定分析。
使用PLINK软件计算两两个体间的Identity by State(IBS)值，得到IBS距离矩阵。并基于IBS距离矩阵，使用PHYLIP软件的邻接法构建系统发育树，最后通过Figtree软件可视化系统发育树。系统发育树结果如图4所示。结果显示，各个猪种可以明显的区分开，可以用于品种鉴定。此外，3个商业猪种(杜洛克猪、大白猪和长白猪)处于一个大的进化枝，而所有中国地方品种处于另一个大的进化枝，北京黑猪位于这两个主要进化枝之间的中间位置，这与北京黑猪的培育历史一致。该结果表明，本发明提供的芯片能够很好地应用于北京黑猪的遗传分类和进化分析，鉴定结果准确可靠。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种用于北京黑猪基因分型的SNP分子标记组合、芯片及其制备方法与应用。本发明用于北京黑猪基因分型的SNP分子标记组合，由49,963个SNP分子标记组成，所述SNP分子标记的核苷酸序列分别如SEQ ID NO：1-49,963所示。应用该分子标记组合可制备北京黑猪分子育种芯片，并可进一步应用于特异性鉴定北京黑猪与其他猪种(欧洲商业猪种、中国地方猪种)、目标性状QTL、关联位点及候选基因鉴定、亲缘关系鉴定和家系划分等方面，具有较好的经济价值和应用前景。

Claims (10)

  1. 用于北京黑猪基因分型的SNP分子标记组合，其特征在于，由49,963个SNP分子标记组成，所述SNP分子标记的核苷酸序列分别如SEQ ID NO：1-49,963所示。
  2. 权利要求1所述的SNP分子标记组合在制备北京黑猪分子育种芯片中的应用。
  3. 一种北京黑猪分子育种50K SNP芯片，其特征在于，包含1-49,963个SNP分子标记，所述SNP分子标记的核苷酸序列分别如SEQ ID NO：1-49,963所示。
  4. 如权利要求1所述的用于北京黑猪基因分型的SNP分子标记组合或权利要求3所述的北京黑猪分子育种50K SNP芯片，其特征在于，所述SNP分子标记的SNP位点位于SEQ ID NO.1-49,963所示的核苷酸序列的第71位。
  5. 一种制备权利要求3所述的北京黑猪分子育种50K SNP芯片的方法，其特征在于，包括以下步骤：
    步骤①：第一类探针的获得：使用中芯一号PLUS猪育种芯片检测北京黑猪群体，对芯片数据进行质控，质控标准为:剔除SNP检出率＜95％、最小等位基因频率＜0.1、哈代-温伯格平衡检验P值＜10 -6以及没有染色体位置信息的SNP位点；将中芯一号PLUS猪育种芯片位置信息从Sus scrofa 10.2版本转为Sus scrofa 11.1版本参考基因组，得到第一类备选SNP；
    步骤②：第二类探针的获得：结合公共数据库NCBI和QTLdatabase获得与猪肉质性状、生长性状、繁殖性状及健康性状相关候选基因内部及上下游SNP位点、全基因组关联分析和QTL结果显著SNP，并基于北京黑猪重测序数据，剔除MAF<0.05的位点，得到第二类备选SNP；
    步骤③：第三类探针的获得：基于北京黑猪、大白猪与长白猪的重测序数据，筛选北京黑猪与大白猪、长白猪的绝对等位基因频率差异均大于等于0.6的位点，并基于北京黑猪重测序数据，剔除MAF<0.05的位点， 得到第三类备选SNP；
    步骤④：第四类探针的获得：使用中芯一号PLUS猪育种芯片检测北京黑猪群体，基于北京黑猪的芯片数据和肉质表型数据进行全基因组关联分析，肉质表型数据包括肌内脂肪、蛋白含量、水分含量、胶原蛋白含量、滴水损失、肉色和屠宰后24小时pH值；由全基因组关联分析鉴定到的与肉质性状显著相关的位点，作为第四类备选SNP；
    步骤⑤：第五类探针的获得：基于北京黑猪重测序数据，保留MAF≥0.1的SNP，得到第五类备选SNP；
    步骤⑥：将常染色体与X染色体分为48kb一个窗口，每个窗口按照第一类至第五类探针的优先级，选择1个SNP填充；
    步骤⑦：将筛选好的SNP位点由Infmium iSelect打分系统打分，将基因内分值<0.7和基因间分值<0.9的位点去掉；对于删掉的不合格的SNP位点，选取距离其最近的SNP位点进行补充，并且再次进行打分，直至全部位点合格，之后将获得的标签序列用于SNP芯片的制作。
  6. 权利要求1所述的用于北京黑猪基因分型的SNP分子标记组合或权利要求3所述的北京黑猪分子育种50K SNP芯片在北京黑猪全基因组关联分析中的应用。
  7. 如权利要求6所述的应用，其特征在于，在进行北京黑猪全基因组关联分析中，应用权利要求3所述的北京黑猪分子育种50K SNP芯片对北京黑猪进行基因型检测，并测定相关表型性状，基于线性混合模型进行全基因组关联分析，线性混合模型的表达式为：
    y＝Xα+Yβ+Zγ+e
    y是表型向量，α为SNP标记效应向量，X是对应于α的关联矩阵，编码为0、1、2，β为非遗传固定效应向量，Y是对应于β的关联矩阵，γ是剩余多基因效应向量，服从正态分布
    Figure PCTCN2022115226-appb-100001
    G是基因组亲缘关系矩阵，
    Figure PCTCN2022115226-appb-100002
    是加性遗传方差，Z是对应于γ的关联矩阵，e为残差向量，服从
    Figure PCTCN2022115226-appb-100003
    I为单位矩阵，
    Figure PCTCN2022115226-appb-100004
    是随机残差。
  8. 权利要求1所述的用于北京黑猪基因分型的SNP分子标记组合或权利要求3所述的北京黑猪分子育种50K SNP芯片在北京黑猪品种鉴定中的应用。
  9. 如权利要求8所述的应用，其特征在于，在进行未知品种的猪的鉴定时，应用权利要求3所述的北京黑猪分子育种50K SNP芯片对所述未知品种的猪进行基因型检测，与已知确定为北京黑猪的芯片数据进行合并，使用PLINK软件对芯片数据质控和计算个体间的IBS距离矩阵，并基于IBS距离矩阵，使用PHYLIP软件的邻接法构建系统发育树，最后通过Figtree软件可视化系统发育树，判断所述未知品种的猪是否为北京黑猪，从而进行品种鉴定。
  10. 权利要求1所述的用于北京黑猪基因分型的SNP分子标记组合或权利要求3所述的北京黑猪分子育种50K SNP芯片在北京黑猪育种中的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117802249A (zh) * 2024-03-01 2024-04-02 中国海洋大学三亚海洋研究院 一种东星斑全基因组snp芯片的制备方法及应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117965760A (zh) * 2024-03-29 2024-05-03 中山大学 一种猪肉质性状选育的snp芯片及其制备方法和应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106164291A (zh) * 2014-03-28 2016-11-23 中国科学院昆明动物研究所 用于在猪中检测产仔数的基因标记的方法和试剂盒
EP3321379A1 (en) * 2016-11-15 2018-05-16 Republic of Korea Genetic marker for determining meat quality traits of pigs and use thereof
WO2019113818A1 (zh) * 2017-12-13 2019-06-20 中国农业大学 猪全基因组50k snp芯片及应用
CN110229914A (zh) * 2019-06-17 2019-09-13 中国农业科学院北京畜牧兽医研究所 一种影响猪肌纤维直径的snp分子标记及其应用
WO2020238218A1 (zh) * 2019-05-30 2020-12-03 浙江大学 嘉兴黑猪及生肉制品的snp标记组合和鉴定方法
CN113981109A (zh) * 2021-12-03 2022-01-28 北京康普森农业科技有限公司 用于猪亲缘关系鉴定的snp分子标记组合、应用及方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106164291A (zh) * 2014-03-28 2016-11-23 中国科学院昆明动物研究所 用于在猪中检测产仔数的基因标记的方法和试剂盒
EP3321379A1 (en) * 2016-11-15 2018-05-16 Republic of Korea Genetic marker for determining meat quality traits of pigs and use thereof
WO2019113818A1 (zh) * 2017-12-13 2019-06-20 中国农业大学 猪全基因组50k snp芯片及应用
WO2020238218A1 (zh) * 2019-05-30 2020-12-03 浙江大学 嘉兴黑猪及生肉制品的snp标记组合和鉴定方法
CN110229914A (zh) * 2019-06-17 2019-09-13 中国农业科学院北京畜牧兽医研究所 一种影响猪肌纤维直径的snp分子标记及其应用
CN113981109A (zh) * 2021-12-03 2022-01-28 北京康普森农业科技有限公司 用于猪亲缘关系鉴定的snp分子标记组合、应用及方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YANG WENJING, LIU ZHEN, ZHAO QIQI, DU HENG, YU JIAN, WANG HONGWEI, LIU XIANCE, LIU HAI, JING XITAO, YANG HONGPING, SHI GUOHUA, ZHO: "Population Genetic Structure and Selection Signature Analysis of Beijing Black Pig", FRONTIERS IN GENETICS, vol. 13, XP093103483, DOI: 10.3389/fgene.2022.860669 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117802249A (zh) * 2024-03-01 2024-04-02 中国海洋大学三亚海洋研究院 一种东星斑全基因组snp芯片的制备方法及应用

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