A pig whole genome low-density 5K SNP chip and its application

Abstract

The present invention relates to the field of molecular marker technology, and specifically discloses a pig whole genome low-density 5K SNP chip and its application. The pig whole genome low-density 5K SNP chip provided by the present invention includes a SNP molecular marker combination for pig genotyping, and the SNP molecular marker combination consists of 5603 SNP molecular markers, and the SNP molecular markers are shown in Table 1. The SNP chip or molecular marker combination provided by the present invention can be applied to multiple fields such as pig whole genome breeding value evaluation, cluster analysis, molecular marker assisted breeding, etc., greatly reducing breeding costs.

Description

Technical Field

[0001] The present invention relates to the field of molecular marker technology, and in particular to a pig whole genome low-density 5K SNP chip and application thereof. Background Art

[0002] SNP (Single Nucleotide Polymorphism) 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. SNP chips are made by fixing fluorescently labeled DNA probes on silicon wafers, and then SNP typing is performed by hybridizing the probe DNA with genomic DNA. SNPs bind to the probes 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.

[0003] Genomic selection (GS) is a strategy that uses high-density SNP chips to obtain marker information within the genome, conduct genetic evaluation and selection for target traits. Compared with traditional breeding methods, genomic selection has many advantages, such as improving the accuracy of breeding value estimation, enhancing selection intensity and accelerating genetic progress. This technology can greatly shorten the generation interval, thereby significantly reducing breeding costs. However, the expensive price of high-density SNP chips is still a major limiting factor for the widespread application of GS in actual livestock production. The design and application of low-density SNP chips is an important method to solve this bottleneck problem. In actual GS applications, low-density chips can be used to first perform genotyping on candidate individuals, and the data measured by low-density chips can be filled into medium and high densities to increase the amount of calculated data, improve selection accuracy, and achieve early selection.

[0004] In 2008, Illumina launched the first gene chip for genomic selection of dairy and beef cattle, the BovineSNP50 chip, which has 54,000 SNPs; two years later, the BovineHD chip containing 777,000 SNP sites was released. In 2011, Illumina's BovineLD chip began to be used in dairy cattle breeding. This low-density chip contains about 7,000 SNPs, which can be filled to 45,000 SNP genotypes and will be used for genomic genetic evaluation. In 2012, Neogen's subsidiary GeneSeek began selling the GeneSeek Genomic Profiler (GGP) cattle low-density SNP chip, which adds an additional 1,800 SNPs to the BovineLD chip site to improve filling accuracy. In 2014, the third version of the GGP cattle low-density SNP chip was released, adding 1,900 additional SNPs compared to the initial version. In 2015, GeneSeek released the 4th version of the GGP cattle low-density SNP chip, which can currently detect more than 30,000 SNPs. These low-density commercial chips have been successfully applied to the breeding practices of dairy and beef cattle, and have performed well in filling and genomic breeding value prediction. In addition, academic studies in dairy cows, beef cattle, pigs, sheep, broilers and laying hens have shown that it is feasible to fill low-density chips to medium and high densities and apply them to GS. Taking the above factors into consideration, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to achieve pig genotyping. The present invention provides a pig whole genome low-density 5K SNP chip, wherein the 5K SNP chip contains a SNP molecular marker combination for pig genotyping.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a SNP molecular marker combination for pig genotyping, which consists of 5603 SNP molecular markers. The SNP molecular markers are shown in Table 1.

[0008] The SNP molecular markers described in the present invention are mainly generated by the following steps: the first step is to screen out the site union of three commonly used pig commercial chips, namely, Porcine SNP 60K (Porcine SNP 60 developed by Illumina), Porcine 80K (Porcine 80k developed by GeneSeek) and CAU50K (KPSISUS50-V1 developed by China Agricultural University and Compson), based on the resequencing data of Large White pigs, and perform routine quality control on these sites (MAF>0.05, hwe>1e-5, call rate>95%) as the original sites for chip design; secondly, the FSFS algorithm was used to cluster sites with linkage disequilibrium (r2) greater than 0.5 into one category, and the sites at the cluster center were selected to be retained, and the rest of the sites were removed to initially reduce the chip density, and then sites with MAF greater than 0.2 were screened to ensure the polymorphism of the sites. Finally, the MOLO algorithm was used to reduce the density to 4,869 sites while ensuring the uniformity and polymorphism of the chip sites; thirdly, other sites with good polymorphism and high detection rate in the resequencing data were added, totaling 734 sites; fourthly, genetic defect sites such as halothane sensitive gene, multiple rib gene, umbilical hernia, inverted nipple, severe immunodeficiency disease, and SNP sites related to important economic traits such as growth rate were added, totaling 12 sites. The final low-density chip contains a total of 5,603 SNP sites.

[0009] In a second aspect, the present invention provides the use of the above-mentioned SNP molecular marker combination in the preparation of a pig whole genome 5K SNP chip, or a pig genome detection reagent or kit.

[0010] In a third aspect, the present invention provides a pig whole genome 5K SNP chip, which comprises 5603 SNP molecular markers, and the SNP molecular markers are shown in Table 1.

[0011] The 5K SNP chip of the present invention adopts the fiber microbead chip made by Illumina's Infinium patented manufacturing technology (US6,429,027). The pig whole genome low-density 5K SNP chip provided by the present invention can be filled with medium and high density chip data, and then used in whole genome selection breeding and other aspects.

[0012] In a fourth aspect, the present invention provides a pig whole genome detection reagent or kit, which comprises 5603 SNP molecular markers, and the SNP molecular markers are shown in Table 1.

[0013] Table 1 Information of 5603 SNP molecular markers

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[0071] In a fifth aspect, the present invention provides the use of the above-mentioned SNP molecular marker combination for pig genotyping or pig whole genome 5KSNP chip or pig whole genome detection reagent or kit in any of the following aspects:

[0072] (1) In pig genotyping, marker-assisted breeding or genome-wide association studies;

[0073] (2) In pig genetic diversity analysis, identification or improvement of economic traits or whole genome selection breeding;

[0074] (3) In cluster analysis, kinship identification or identification of pig breeds.

[0075] It is also within the protection scope of the present invention to select one or more molecular markers from the SNP molecular marker combination shown in Table 1 of the present invention for use alone or in combination.

[0076] In a sixth aspect, the present invention provides the use of the above-mentioned pig whole genome 5K SNP chip in filling medium and high density chips.

[0077] In the seventh aspect, the present invention provides a SNP molecular marker combination for detecting genetic defects or important economic traits, which is composed of 12 SNP molecular markers (each locus is associated with a genetic defect or important economic trait of a pig), and the SNP molecular markers are the SNP molecular markers numbered 1554, 1763, 1833, 1923, 2115, 2641, 3035, 3036, 3388, 3982, 3989 and 5463 in Table 1.

[0078] The beneficial effects of the present invention are at least:

[0079] The SNP marker sites provided by the present invention are evenly distributed on the 18 autosomes of the pig genome (excluding sex chromosomes), and can be filled into the original sites using family information, with little loss of genotype information. The filled data can be used to carry out genome selection to obtain acceptable evaluation accuracy.

[0080] The SNP marker sites provided by the present invention have a high MAF in the test population, so compared with other low-density chips, they have higher information entropy and genotype filling accuracy. The average MAF of the original sites in the test population is 0.29, while the MAF of the 5,603 SNP sites included in the present invention in the test population reaches 0.36.

[0081] The chip prepared with the SNP molecular marker combination of the present invention can be mainly used in pig genome selection technology and basic research in the field of molecular genetics. This chip contains a small number of sites, but the amount of information contained in the sites is large, and the population size referenced during the design process is large, with high reliability and universality. Therefore, more individuals can be measured at a very low cost, the amount of genotype data can be increased, the accuracy of selection can be improved, and the realization of early selection can be promoted. Through the evaluation of whole genome breeding values, important production performance can be measured early, the best can be selected, the generation interval can be shortened, and genetic progress can be accelerated. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0083] Figure 1 It is a schematic diagram of the research and development process of the present invention.

[0084] Figure 2 This is a distribution map of the SNP sites of the present invention within the entire genome.

[0085] Figure 3 The distance between adjacent SNPs of the SNP molecular markers used for preparing the pig whole genome 5K SNP chip of the present invention (unit: Mb).

[0086] Figure 4 This is a statistical diagram of the distribution of SNP sites in the whole genome. The bar graph represents the chromosome length, and the line graph represents the proportion of SNP sites on the chromosome.

[0087] Figure 5 This is the clustering result of principal component analysis of the five local pig breeds in Example 4 of the present invention. DETAILED DESCRIPTION

[0088] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0089] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the equipment and reagents used in each example are conventionally commercially available. Unless 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 the conditions recommended by the manufacturer's instructions.

[0090] Example 1 Acquisition of SNP Molecular Markers and Preparation of 3K Pig Whole Genome SNP Chip

[0091] SNP molecular markers are obtained through the following four steps.

[0092] Step 1: Use resequencing technology to detect the genotypes of 19,548 large white pigs in Chifeng pig farms, and extract the loci of pig 60K (PorcineSNP60 developed by Illumina), pig 80K (Porcine80k developed by GeneSeek) and CAU 50K (KPSISUS50-V1 developed by China Agricultural University and Compson), totaling 93,577 loci. Use PLINK software to perform quality control on these genotype data, individual call rate>95%, MAF>0.05, Hardy-Weinberg equilibrium test>1e-5, remove the loci on the sex chromosomes and the loci with unknown physical positions, and the remaining 37,305 loci (hereinafter referred to as "original loci") are used as the original loci library for chip design.

[0093] Step 2: First, the FSFS algorithm was used to cluster sites with linkage disequilibrium (r2) greater than 0.5 into one category, and the site at the cluster center was retained as the characteristic of this category. The remaining sites were removed to initially reduce the chip density, and then sites with MAF greater than 0.2 were screened to ensure the polymorphism of the sites. Finally, the MOLO algorithm was used to reduce the density to 4,869 sites while ensuring the uniformity and polymorphism of the chip sites.

[0094] Step 3: To ensure the uniformity of the chip sites, additional sites with good polymorphism and high detection rate in the resequencing data were added, totaling 734 sites.

[0095] Step 4: 12 functional sites were added, which were respectively related to genetic defects such as halothane sensitivity gene, multiple rib gene, umbilical hernia, inverted nipple, severe immunodeficiency disease and muscle synthesis ability. The numbers of the functional sites are 1554, 1763, 1833, 1923, 2115, 2641, 3035, 3036, 3388, 3982, 3989, 5463 in Table 1.

[0096] The identified 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 to supplement and score again. According to the above steps, 5,603 tag SNP sites were finally obtained after identification and screening. According to the design requirements of Illumina Infmium iSelect HD, 5,603 types of microbeads are required. The specific biological polymorphisms of all 5,603 tag SNP sites are recorded in Table 1. These tag sequences were submitted to Illumina for design and production of Infmium SNP chips.

[0097] The 5K SNP chip consisting of the above 5,603 tag SNP loci has the following advantages:

[0098] The low-density chip has low cost, contains a small number of sites but high quality and a large amount of information, and can measure more individuals at a very low cost, thereby increasing the amount of data available in the genome selection process and improving the accuracy of the assessment. The SNP molecular markers provided by the present invention have the characteristics of uniform distribution and high whole-genome coverage, and have high genotype filling accuracy. Family information can be used to fill to medium and high density using software, and then used for genome selection, thereby achieving early seed selection, shortening generation intervals, and accelerating genetic progress. The SNP molecular markers provided by the present invention can also be used as one of the bases for variety identification.

[0099] The schematic diagram of the research and development process of the present invention is shown in Figure 1 The distribution of the chip sites of the present invention on each chromosome is shown in Table 2. The comparison of the parameters of the chip of the present invention (5K low density) and the original site is shown in Table 3. The distribution of SNP sites in the whole genome is shown in Figure 2 The distance between adjacent SNPs in SNP molecular markers is shown in Figure 3 The statistical diagram of SNP loci distribution at the whole genome level is shown in Figure 4The genome version number used in the present invention is pig (sus scrofa) version 11.1.

[0100] Table 2 Distribution of the number of sites on the chip of the present invention on each chromosome

[0101] chromosome Number of SNP sites chromosome Number of SNP sites chromosome Number of SNP sites 1 675 7 300 13 516 2 377 8 343 14 349 3 327 9 341 15 345 4 336 10 172 16 193 5 266 11 203 17 151 6 420 12 152 18 137

[0102] Table 3 Comparison of site parameters between the chip of the present invention (5K low density) and the original

[0103] Number of sites MAF Mean Average interval (kb) 5K Chip 3,627 0.36 403 Original site 37,305 0.29 61

[0104] Example 2 Application of the 5K pig whole genome SNP chip of the present invention in high-density chip filling in pigs

[0105] The original loci designed by the present invention comprising 37,305 loci described in Example 1 and the 5K chip described in Example 1 of the present invention were used to perform genotype detection on Large White pigs in two pig farms in Chifeng and Beijing. After the obtained genotype data were quality controlled, the 5K low-density chip data were filled with genotypes. The specific steps are as follows:

[0106] (1) DNA resequencing data of Large White pigs from two pig farms were used. Among them, 7,808 individuals born in 2022 in the Chifeng farm were used as the validation group, retaining 5K low-density chip loci and 37,305 original loci; 11,397 individuals born from 2018 to 2021 were used as the reference group, retaining only 37,305 original loci. 1,800 individuals born from 2021 to 2022 in the Beijing farm were used as the validation group, retaining 5K low-density chip loci and 37,305 original loci; 2,487 individuals born from 2018 to 2020 were used as the reference group, retaining only 37,305 original loci.

[0107] (2) Using the reference group information and Beagle software, the 5K chip data of the validation group individuals in the Chifeng and Beijing pig farms were filled into the original site data of 37,305 sites, and compared with their real original site data, the proportion of the correctly filled alleles to the total alleles that need to be filled was calculated as the accuracy evaluation index of the genotype filling. In this embodiment, the percentage of correct filling was calculated separately for the two alleles of each marker. The accuracy calculated by this method is called the allele accuracy. The final calculated accuracies were 98.71% and 91.76%, respectively.

[0108] Example 3 Application of the 5K pig whole genome SNP chip of the present invention in genomic selection breeding

[0109] This example uses the real original site data recorded in Example 2 and the data filled into the original site using the 5K low-density chip of the present invention to perform genetic evaluation. Large white pigs from two pig farms in Chifeng and Beijing were tested, and the "GBLUP" and "ssGBLUP" methods were used to perform genetic evaluation on age (AGE) / back fat thickness (BF) up to 100 kg body weight. The specific steps are as follows:

[0110] (1) As described in Example 2, DNA of Large White pigs from two pig farms was extracted for SNP chip detection.

[0111] (2) The division criteria are as described in Example 2, with birth time as the criterion for dividing the reference group and the validation group, but in this example, only individuals with both genotype and phenotype are retained as the objects of genome evaluation. 1,656 individuals born in 2022 in the Chifeng field were used as the validation group, and 4,226 individuals born in 2018-2021 were used as the reference group; 950 individuals born in 2021-2022 in the Beijing field were used as the validation group, and 2,487 individuals born in 2018-2020 were used as the reference group. The reference group uses genotype information of 37,305 original sites, and the validation group uses 37,305 original sites and 5K chips filled into the original sites to provide genotype information. The double-trait GBLUP and ssGBLUP models were used to evaluate the 100kg body weight age (AGE) / backfat thickness (BF) traits of Large White pigs from two pig farms in Chifeng and Beijing (the number of individuals in the Beijing pig farm was small and the solution could not be obtained using GBLUP alone), and the DMU software was used for genetic evaluation.

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[0113] where y1 and y2 are the phenotypic observation values ​​of AGE and BF traits, μ1 and μ2 are the mean values ​​of AGE and BF traits, g1 and g2 are the additive breeding values ​​of AGE and BF traits, and e1 and e2 are the residuals of AGE and BF traits.

[0114] The accuracy of genetic breeding value estimation was measured by the correlation between the genomic estimated breeding value (GEBV) and the corrected phenotype, where the corrected phenotype was obtained by adding the estimated breeding value (EBV) calculated by the pedigree-based BLUP model and the estimated residual; unbiasedness was the regression coefficient of the corrected phenotype on the genomic estimated breeding value.

[0115] (3) Results: The specific evaluation accuracy and unbiasedness are shown in Tables 4 and 5.

[0116] Table 4 Genetic evaluation accuracy of different chips

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[0118] Table 5 Unbiasedness of genetic evaluation of different chips

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[0120] Example 4 Principal component analysis of five Chinese local pig breeds based on the 5K whole genome SNP chip of the present invention

[0121] The 5K pig whole genome SNP chip provided in Example 1 of the present invention was used to perform genotype detection on DNA extracted from 15 Xiangcun black pigs, 15 Yacha pigs, 15 Tibetan pigs, 15 Liangshan black pigs, and 15 Yantai black pigs. Principal component analysis (PCA) was performed using GCTA software, and the first and second principal components were plotted. The results are as follows: Figure 5 The results showed that the five local pig breeds could be clearly distinguished, among which only the Xiangcun black pig and Liangshan black pig groups were slightly mixed, which showed that this low-density chip had a certain role in identifying and distinguishing pig breeds and could be used as one of the reference standards for pig breed identification.

[0122] Example 5 Application of the 5K Whole Genome SNP Chip of the Present Invention in Other Aspects

[0123] The 5K pig whole genome SNP chip provided in Example 1 of the present invention is used to screen and identify the main effect genes of important economic traits in the pig population. Specifically as follows: First, the SNP chip of the present invention is used to perform genotype detection on the pig population, and combined with the phenotypic data of the pig target traits recorded in actual production, a whole genome association analysis is performed, and according to the results of the significance test, the SNP sites significantly associated with the target traits are selected. The functional genes within 500kb upstream and downstream of the significant SNP are annotated as candidate genes, and the gene functions are queried in combination with the existing database, so as to achieve the purpose of locating and mining the target trait genes, and provide theoretical support for subsequent molecular verification.

[0124] The 5K pig whole genome SNP chip provided in Example 1 of the present invention is used for kinship identification. Specifically, the SNP chip of the present invention is used to detect the genotype of the target individual, and then the genotype information is used to establish a kinship matrix, and the kinship is identified based on the kinship coefficient between individuals combined with pedigree records (for example, the kinship coefficient between parents and children and full siblings is about 0.5, and the kinship coefficient between half siblings is about 0.25). In addition, based on the results of the kinship matrix, the calculation of the population inbreeding coefficient, pedigree error detection, etc. can be performed later.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A combination of SNP molecular markers for pig genotyping, characterized in that: It consists of 5603 SNP molecular markers, and the SNP molecular markers are shown in Table 1.

2. Use of the SNP molecular marker combination according to claim 1 in the preparation of a pig whole genome 5K SNP chip, or a pig genome detection reagent or kit.

3. A pig whole genome 5K SNP chip, characterized in that: It contains 5603 SNP molecular markers, and the SNP molecular markers are shown in Table 1.

4. A pig whole genome detection reagent or kit, characterized in that: It contains 5603 SNP molecular markers, and the SNP molecular markers are shown in Table 1.

5. Use of the SNP molecular marker combination for pig genotyping according to claim 1, the pig whole genome 5K SNP chip according to claim 3, or the pig whole genome detection reagent or kit according to claim 4 in pig genotyping detection, molecular marker-assisted breeding or whole genome association analysis.

6. Use of the SNP molecular marker combination for pig genotyping according to claim 1, the pig whole genome 5K SNP chip according to claim 3, or the pig whole genome detection reagent or kit according to claim 4 in pig genetic diversity analysis, economic trait identification or improvement, or whole genome selection breeding.

7. Use of the SNP molecular marker combination for pig genotyping according to claim 1, the pig whole genome 5K SNP chip according to claim 3, or the pig whole genome detection reagent or kit according to claim 4 in pig cluster analysis, kinship identification, or identification of pig breeds.

8. Use of the porcine whole genome 5K SNP chip according to claim 3 in filling medium and high density chips.

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