Pig whole genome low-density 6K SNP chip and application thereof

Abstract

The invention discloses a pig whole genome low-density 6K SNP (Single Nucleotide Polymorphism) chip, and the chip contains 5,866 SNP loci. The SNP chip provided by the invention is suitable for a plurality of fields of pig whole genome association analysis, whole genome selective breeding, population genetics analysis, character-related candidate gene and QTL site analysis and positioning and the like, and has relatively high value in production application and scientific research.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology, bioinformatics and genome breeding technology, and in particular to a pig low-density 6K SNP chip and applications thereof. Background Art

[0002] Single nucleotide polymorphisms (SNPs) are molecular markers formed at the genomic level by single-base deletions, insertions, transitions, and transversions. SNPs are numerous, widely distributed, amenable to rapid, large-scale screening, and convenient for genotyping. As important molecular genetic markers, SNPs contribute to the genetic variation of complex traits and are relatively easy to automate and scale using gene chip technology, leading to their widespread application in genomic breeding technology.

[0003] Genomic selection (GS) is a modern breeding technique that leverages genomic information and computational technology to assist in breeding, achieving more efficient and accurate selection. Genomic selection utilizes single nucleotide polymorphisms (SNPs) as molecular markers to analyze the genetic background and genomic variation of candidate animals, assess and predict phenotypic traits, and optimize breeding strategies through computer simulation and analysis. Compared to traditional breeding methods, genomic selection allows for earlier selection of animals, thereby shortening generation intervals and accelerating the breeding process.

[0004] During genomic selection, candidate animals must be subjected to SNP genotyping. Currently, the primary method is SNP microarray testing. In the field of porcine genomic selection, SNP microarrays with SNP densities above 50K are currently the primary method used. However, these chips are expensive and difficult to implement in production practice.

[0005] Therefore, it is necessary to develop a whole-genome low-density SNP chip suitable for pigs to meet market demand. Summary of the Invention

[0006] In order to solve one of the above problems existing in the prior art, the present invention provides a pig whole genome low-density SNP chip and its application.

[0007] In a first aspect, the present invention provides a SNP molecular marker for the whole pig genome, wherein the SNP molecular marker is selected from at least one of the markers shown in Table 4.

[0008] In a second aspect, the present invention provides a molecular probe for specifically identifying the SNP molecular marker described in the first aspect.

[0009] In a third aspect, the present invention provides a pig whole-genome SNP chip, wherein the SNP chip comprises probes targeting the SNP molecular markers described in the first aspect.

[0010] In some embodiments, the porcine whole-genome SNP chip of the present invention contains 5,866 SNP sites.

[0011] In some embodiments, the molecular marker is associated with one or more of pig production traits and economic traits.

[0012] In some embodiments, the production traits include one or more of reproduction, birth weight, daily weight gain, litter size, intramuscular fat content, and growth rate.

[0013] In some embodiments, the economic traits include one or more of meat yield, meat quality, feed conversion rate, disease resistance, for example, corrected age up to 100 kg (AGE), corrected backfat thickness up to 100 kg (BF), etc.

[0014] In a fourth aspect, the present invention provides a kit comprising the molecular probe described in the second aspect, or the SNP chip described in the third aspect.

[0015] In a fifth aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in pig genotyping detection.

[0016] In some embodiments, the SNP markers on the SNP chip of the present invention are fixed by microarray technology, and high-throughput and high-precision genotyping can be achieved using hybridization and fluorescence detection technology.

[0017] In some embodiments, the SNP chip of the present invention can achieve efficient acquisition and integration of genetic analysis data, and achieve efficient management and utilization of data by constructing a comprehensive database.

[0018] In a sixth aspect, the present invention provides the use of the molecular probe, or the SNP chip, or the kit in whole genome breeding of pigs or early selection of pig production traits.

[0019] In some embodiments, the application in pig whole genome breeding includes using the SNP chip of the present invention to fill medium- and high-density chips of pigs.

[0020] In some embodiments, the SNP chip of the present invention establishes a breeding value prediction model by performing high-density genotyping on individual pigs and combining phenotypic data.

[0021] In some embodiments, the SNP chip of the present invention establishes a breeding value prediction model by genotyping a large number of pig individuals and combining phenotypic data with environmental factors.

[0022] In some embodiments, the SNP chip of the present invention utilizes SNP markers for genomic background selection.

[0023] In some embodiments, the SNP array of the present invention expands genome-wide association studies by increasing the density of SNP markers and identifying gene loci associated with economically important traits.

[0024] In some embodiments, the SNP chip of the present invention can be used for early selection of target traits by selecting SNP markers associated with important economic traits.

[0025] In some embodiments, the SNP chip of the present invention can be used to perform early selection using SNP markers, and can be used to screen sows before they exhibit target traits, thereby determining the breeding direction in advance.

[0026] In some embodiments, the SNP chip of the present invention utilizes genome-wide selection to optimize breeding strategies according to different breeding goals.

[0027] In some embodiments, the use of whole genome selection can optimize breeding strategies according to different breeding goals, such as increasing meat yield, improving meat quality, enhancing disease resistance, etc.

[0028] In a seventh aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in pig breed identification.

[0029] In some embodiments, the SNP chip of the present invention can construct genomic fingerprints of different pig breeds by performing genotyping on different pig breeds, thereby accurately identifying the breed origin of the pigs.

[0030] In an eighth aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in pig kinship identification.

[0031] In a ninth aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in analyzing genetic diversity of pig populations.

[0032] In some embodiments, the SNP chip of the present invention can be used to assess the genetic structure and variation level of a pig population through genetic diversity analysis.

[0033] In a tenth aspect, the present invention provides the use of the molecular probe, or the SNP chip, or the kit in the positioning analysis of target trait QTL (quantitative trait locus).

[0034] Compared with the prior art, the present invention has at least one of the following advantages:

[0035] The pig genome-wide low-density 6K SNP chip provided by this invention can rapidly and cost-effectively detect relevant SNP markers, making it possible to popularize early genomic selection in pigs and significantly improving pig breeding progress in my country. The SNP molecular marker combination provided by this invention features uniform SNP loci distribution and high genome-wide coverage. Using these SNP markers for genome-wide breeding effectively enhances the degree of association with traits through linkage disequilibrium (LD), ensuring the accuracy of breeding value estimates.

[0036] The porcine genome-wide, low-density 6K SNP chip provided by this invention features selected SNP sites highly correlated with genes associated with important economic traits in pigs, enabling early selection and gene mapping for target traits. This SNP chip improves breeding accuracy while providing strong support for subsequent molecular validation and gene function studies, demonstrating its high value in both production applications and scientific research.

[0037] The inventors of the present application have screened, discovered and identified for the first time a group of key sites in the 6K SNP chip with stable genetic effects across generations, breaking through the limitations of traditional chips that rely solely on single-group association analysis. The 6K SNP chip of the present application not only covers the basic production traits and economic traits of pigs such as meat production, meat quality, and disease resistance through dynamically screened SNP markers, but also incorporates important production traits and economic traits such as birth weight, daily weight gain, litter size, intramuscular fat content, growth rate, backfat thickness, and feed conversion rate into the detection system. By adding these newly discovered related sites, the 6K SNP chip provides more comprehensive genetic information and can more accurately map quantitative trait loci (QTL), thereby improving the accuracy of genomic selection. This helps to optimize breeding decisions, especially in whole-genome breeding and early trait selection of pigs, and improves the reliability of genetic evaluation and breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a genome-wide distribution diagram of the sites of the pig whole-genome low-density 6K SNP chip of the present invention.

[0039] Figure 2 Schematic diagram of the proportion of sites in chromosomes of the pig whole genome low-density 6K SNP chip of the present invention.

[0040] Figure 3 Schematic diagram of the MAF levels of the sites on the pig whole genome low-density 6K SNP chip of the present invention.

[0041] Figure 4 This is a schematic diagram of the distance between adjacent SNPs in the SNP molecular markers of the pig whole genome low-density 6K SNP chip of the present invention.

[0042] Figure 5 Schematic diagram of the clustering results of principal component analysis of four breeding pig groups in Example 3 of the present invention. DETAILED DESCRIPTION

[0043] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0044] definition

[0045] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0046] Unless the context clearly indicates otherwise, references herein that do not explicitly state a quantity include the plural form thereof. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art.

[0047] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0048] The following examples and accompanying drawings are provided to facilitate understanding of the present invention. However, it should be understood that these examples and accompanying drawings are intended to illustrate the present invention only and are not intended to limit the present invention in any way. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present invention.

[0049] The technical solution of the present invention is not limited to the specific embodiments, and all technical variations made based on the technical solution of the present invention fall within the protection scope of the present invention.

[0050] Example 1. Preparation of a low-density 6K SNP chip for the entire pig genome

[0051] (1) Construction of high-quality SNP locus dataset

[0052] 1,270 Duroc pigs, 1,475 Landrace pigs, and 4,173 Large White pigs were selected from great-grandparent breeding farm A, and 5,037 Large White pigs were selected from breeding farm B. The genotype detection chip model KPSISUS50-V1 (CAU 50K) was used for testing.

[0053] The raw data from the four populations were quality-controlled separately, using the same quality control criteria as the conventional genomic selection genotyping process used in production. Parameters were set as follows: MAF (minimum allele frequency) > 0.05, HWE (Hardy-Weinberg equilibrium) > 1e-5, and call rate (genotyping detection rate) > 95%. The individual detection rate was controlled to be > 95%. All sex chromosome loci were removed, leaving only loci on 18 pairs of autosomes. This resulted in four population SNP datasets containing 25,454, 30,681, 31,945, and 32,049 SNPs, respectively. The high-quality loci from these four populations were intersected, and the resulting 18,365 SNPs were used as the preliminary SNP dataset for low-density array design.

[0054] (2) Preliminary SNP site heterozygosity score

[0055] The sum of the heterozygosity of the SNP sites in the above four populations is defined as the heterozygosity score (hetscore), and is calculated using the following formula:

[0056]

[0057] where p ij is the minimum allele frequency (MAF) of the ith SNP in the jth variety.

[0058] (3) Sliding window uniformly screens 6K SNP chip sites

[0059] To ensure that the designed low-density array is evenly distributed with the original loci, a sliding window is set on each of the 18 autosomes in the genome, and the loci with the highest hetscore within each window are selected for inclusion in the candidate SNP dataset. The number of loci selected on a specific chromosome and the sliding window length are set according to the following formula:

[0060] num i =num_6k all / num_raw all *num_raw i

[0061] Among them, num i is the number of sites on chromosome i on the low-density chip, num_6k all is the total number of sites on the 6K low-density chip, num_raw all The total number of original sites in the chip design, num_raw i The original number of sites designed for the chip on chromosome i;

[0062] len_window i =len_chr i / num_chr i

[0063] Among them, len_window i is the window length on chromosome i, len_chr i is the length of chromosome i, num_chr i is the number of sites on chromosome i in the low-density chip.

[0064] In order to ensure the accuracy of genotype filling and provide sufficient flanking information, the first and last SNP sites on each chromosome were retained. A total of 5,866 SNP sites were screened and their distribution across the whole genome was shown in the figure below. Figure 1 The specific number of microarray sites distributed on each chromosome is shown in Table 1.

[0065] The 5,866 SNP sites screened are evenly distributed across chromosomes, such as Figure 2 The average MAF value of the selected sites is shown in Figure 3 The average spacing of the selected SNPs is shown in Figure 4 shown.

[0066] Table 1. The number of SNP chip sites in the whole pig genome distributed on each chromosome

[0067]

[0068] Example 2. Process of detecting pig DNA samples using a pig whole genome low-density 6K SNP chip

[0069] (1) Sample collection and preservation

[0070] For microarray testing, samples should be pig ear or tail root tissue. The sample volume should be soybean-sized. Before collection, disinfect the sampling site with iodine and 75% ethanol. After sample collection, immediately soak the sample in 75% ethanol and store it at -20°C.

[0071] (2) DNA extraction gene

[0072] Pig genomic DNA extraction is performed using a genomic DNA extraction kit. The specific process is adjusted according to the sampling type, whether it is ear margin tissue or tail root tissue. The extracted genomic DNA should be stored at -20°C. DNA quality is verified using 1% agarose gel electrophoresis and a micro-spectrophotometer. Qualified samples have clear main bands, a total DNA (no RNA) content ≥1.0 μg, a concentration ≥20 ng / μL, a volume >50 μL, and an OD value of 1.8 ≤ 260 :OD 280 ≤2.0, OD 260 :OD 230 ≥1.8, no degradation or slight degradation.

[0073] (3) Pig DNA library construction and chip detection

[0074] The pig genomic DNA sample is denatured according to the chip hybridization reaction pre-treatment steps, and then hybridized with the probes on the chip microbeads. The unhybridized DNA is then 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 immune combination of the label and the fluorescent group, and the final genotyping result is generated by the chip reader.

[0075] Example 3. Principal component analysis based on the pig genome-wide low-density 6K SNP chip of the present invention

[0076] The porcine genome-wide low-density 6K SNP chip provided by the present invention was used to perform DNA genotyping on the Large White, Landrace, and Duroc pig populations of Ranch A. A total of 225 individuals were tested, including 33 Duroc pigs, 38 Landrace pigs, and 154 Large White pigs. After preliminary quality control filtering of the raw data, principal component analysis (PCA) was performed using PLINK software. The first two principal components of each individual were calculated and plotted. The results are shown in Figure 2. Figure 5 The results show that PC1 and PC2 can achieve breed resolution of 46.76% and 33.62%, respectively. Duroc (DD), Landrace (LL), and Large White (YY) individuals, represented by green, blue, and red dots, respectively, are clustered together and significantly separated from each other. These principal component analysis results demonstrate the applicability of the SNP chip of the present invention for pig breed identification, providing accurate and reliable results.

[0077] Example 4. Genetic evaluation based on the pig genome-wide low-density 6K SNP chip of the present invention

[0078] DNA genotyping was performed on 580 Large White pigs born between 2019 and 2020 at Ranch B using the whole-genome low-density 6K SNP array provided by this invention. The resulting raw genotype data were first quality-controlled and filtered. Then, using Beagle software, genotype imputation was performed using a reference population of 4544 Large White pigs born between 2017 and 2022 that had been tested using the CAU 50K array. The genotype accuracy of the imputed 600 Large White pigs was 95.92%, as shown in Table 2.

[0079] Table 2. Accuracy of the pig whole genome SNP chip filled to CAU 50K

[0080]

[0081] Subsequently, combined with phenotypic data from the above populations at Farm B, genetic evaluation was performed using DMU software to calculate genomic breeding values and assess the accuracy of the genetic evaluation. The traits evaluated were age (age) corrected to 100 kg and backfat (BF) corrected to 100 kg. A dual-trait animal model was established using the "GBLUP" method. Evaluations were performed using two genotype datasets, one based on the original 50K loci and one based on the 6K microarray filled to 50K loci. The results are shown in Table 3.

[0082] Table 3. Accuracy of the pig whole genome SNP chip filled to CAU 50K

[0083]

[0084] As shown in Table 3, for the two growth phenotypes of age (AGE) corrected to 100 kg and backfat (BF) corrected to 100 kg, the accuracy of genetic evaluation using a 6K chip and then populated into a 50K locus dataset was very close to that of the original 50K locus dataset. These results demonstrate that the present invention, after genotype data population, can achieve performance similar to that of a 50K chip, offering a higher cost-effectiveness, making it suitable for early genetic evaluation in pigs and facilitating widespread adoption.

[0085] Example 5. Application of 6K SNP Chip to Mining Genes Related to Important Economic Traits of Breeding Pigs

[0086] The pig whole genome low-density 6K SNP chip provided by the present invention was used to perform DNA genotyping on a pig population. The obtained genotypic data were combined with the phenotypic data of pig birth weight, daily weight gain, litter size, meat yield, meat quality, disease resistance, age corrected to 100 kg, backfat thickness corrected to 100 kg, growth rate, feed conversion rate and other traits recorded in actual production. GWAS analysis was performed using GEMMA software. Based on the PCA results, a single marker regression model was used to perform the following:

[0087] y=Xm+Wa+e

[0088] Where y represents the phenotypic value studied in this example, m represents the SNP marker effect vector, a represents the residual polygenic effect vector following a normal distribution, X and W are the occurrence matrices of the response variables associated with m and a, respectively, and e represents the residual error vector following a normal distribution. Based on the results of the significance test, SNP sites significantly associated with the target trait were selected, and the annotated functional genes were considered candidate genes within a range of 500 kb upstream and downstream of the significantly associated SNP sites.

[0089] Example 6. Application of 6K SNP Chip in Identifying the Relationship of Breeding Pigs

[0090] The pig population was tested for DNA genotype using the pig genome-wide low-density 6K SNP chip provided by the present invention. The obtained genotype data were first used to calculate the genetic distance between individual pigs using the allele-based dissimilarity (IBS) of the PLINK software. The calculated results were used to construct a phylogenetic tree using the neighbor-joining method (NJ) in the MEGA software. At the same time, the genotype information was used to establish a kinship matrix, in which the kinship coefficient between parents and children and full siblings was approximately 0.5, and the kinship coefficient between half siblings was approximately 0.25. Combining the phylogenetic tree and the kinship matrix results, the kinship of the pigs can be determined and the pedigree correction can be achieved.

[0091] Example 7. Chip Sites

[0092] The porcine whole-genome 6K SNP chip designed by the present invention contains 5,866 SNP sites. The position information of all SNP sites is shown in Table 4.

[0093] Table 4. Location information of SNP sites

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A SNP molecular marker for the whole pig genome, characterized in that: The SNP molecular marker is selected from at least one of the markers shown in Table 4.

2. A molecular probe, characterized in that The molecular probe is used to specifically identify the SNP molecular marker according to claim 1.

3. A pig whole genome SNP chip, characterized in that: The SNP chip comprises probes targeting the SNP molecular markers according to claim 1.

4. The pig whole genome SNP chip according to claim 3, characterized in that The molecular marker is associated with one or more of pig production traits and economic traits; Preferably, the production traits include one or more of reproduction, birth weight, daily weight gain, litter size, intramuscular fat content, and growth rate; Preferably, the economic traits include one or more of meat yield, meat quality, feed conversion rate, and disease resistance.

5. A kit, characterized in that The kit comprises the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4.

6. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in pig genotyping detection.

7. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in whole genome breeding of pigs or early selection of pig production traits.

8. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in pig breed identification.

9. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in pig kinship identification.

10. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in analysis of genetic diversity of pig populations or QTL mapping analysis of target traits.

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