A 17k snp sequencing typing chip for breeding of pig daily age traits of over 100 kg body weight and application

Abstract

The present application relates to a kind of pig 17K SNP sequencing typing chip of hundred kilogram body weight day age character breeding.The present application is obtained by using the genetic marker specific screening of large-scale low-depth whole genome resequencing technology and the functional marker information associated with pig hundred kilogram body weight day age character, greatly eliminates the noise site in sequencing data information, while retaining the functional site associated with pig hundred kilogram body weight day age character body weight, finally forms a kind of 17K SNP sequencing typing chip applied to pig hundred kilogram body weight day age character breeding, reduces the typing cost, while greatly improves the breeding accuracy of pig hundred kilogram body weight day age character.

Description

Technical Field

[0001] This invention relates to the field of genome breeding, specifically to a 17KSNP sequencing and genotyping chip for breeding traits in pigs up to 100 kg in weight and at a given age, and its application. Background Technology

[0002] In 2001, Theo Meuwissen et al. first proposed the innovative concept of Genomic Selection (GS) technology. This is another innovative technology since the implementation of BLUP breeding technology in the last century. It uses high-density markers covering the entire genome for selective breeding. It accelerates genetic progress by ① shortening the generation interval through early selection and ② improving the accuracy of Genomic Estimated Breeding Value (GEBV) estimation. It has a good predictive effect, especially for complex traits with low heritability and difficult measurement. It truly applies genomic technology to breeding practice and is currently driving revolutionary progress in plant and animal breeding. It is one of the most important and cutting-edge common technologies in modern seed industry.

[0003] Single nucleotide polymorphisms (SNPs), as mainstream genetic markers, are numerous, widely distributed, and genetically stable in the genome. They are widely used in human and animal research for elucidating the genetic mechanisms of various traits, selective evolution studies, and genomic selection. In genomic selection applications, high-throughput SNP analysis over the past decade has primarily relied on SNP microarray technology. However, traditional solid-phase SNP microarrays suffer from several drawbacks, including: ① fixed markers that cannot be expanded; ② poor universality across different populations; ③ significant differences in breeding effects between different phenotypes; and ④ high cost. These issues limit the large-scale application of genome-wide selection technology in breeding. In recent years, sequencing-based SNP genotyping methods have developed rapidly. A representative example is the so-called "liquid-phase microarray" method, whose core principle is to perform targeted enrichment sequencing of target regions of the genome to genotype SNPs at thousands to tens of thousands of loci. Although this technology is more flexible in terms of site design than traditional solid-phase chips, the significant differences in the targeting of sequencing at different locations in the genome mean that its analysis and usage costs cannot be effectively reduced in principle, thus limiting its large-scale breeding applications.

[0004] In pig farming, growth traits are among the most important economic traits. The quality of growth traits is directly related to the economic benefits of pig farms, and genetic improvement can enhance the overall economic efficiency of pig herds. The age-to-100 kg weight trait is one of the important traits for measuring pig growth rate; this phenotype is obtained by adjusting for actual body weight and age. Overall, this trait remains a typical quantitative trait, and rapid breeding for the age-to-100 kg weight trait still requires genomic selection technology. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a 17K SNP sequencing genotyping chip for breeding pigs at 100 kg body weight age. This invention utilizes large-scale low-depth whole-genome resequencing technology to specifically screen for functional markers associated with the 100 kg body weight age trait in pigs. This significantly reduces noise sites in the sequencing data while retaining functional sites related to body weight, ultimately resulting in a 17K SNP sequencing genotyping chip for breeding pigs at 100 kg body weight age. This reduces genotyping costs while greatly improving the accuracy of breeding for the 100 kg body weight age trait.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A 17K SNP sequencing and genotyping chip for breeding traits in pigs at 100 kg body weight and age, characterized by containing 16,932 SNP loci.

[0008] The SNP sites in the sequencing genotyping chips described above are shown in Table A:

[0009] Table A. SNP sites in the genotyping chip

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[0027] Another objective of this invention is to provide a method for fabricating a 17K SNP sequencing and genotyping chip for breeding pigs at 100 kg body weight and age.

[0028] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0029] A method for fabricating a 17K SNP sequencing and genotyping chip for breeding pigs at 100 kg body weight and age, characterized by comprising the following steps:

[0030] Step 1: Perform low-depth resequencing of the genomic DNA of each individual in the pig sample population, with a sequencing depth of 0.4×-0.8× / sample;

[0031] Step 2: Based on the sequencing data obtained in Step 1, perform genomic SNP marker detection to identify polymorphic sites in the above-mentioned pig sample population, and perform genotyping on the polymorphic sites to obtain whole-genome SNPs;

[0032] Step 3: Weigh individuals in the pig population within the range of 85-130kg, and correct them according to the following formula (1) to obtain the age-to-weight phenotype at 100kg, where the value A depends on the breed and sex, and outliers are removed;

[0033] Target weight age = Actual age + (Target weight - Actual weight) × ;

[0034] Step 4: Randomly select a fixed number of individuals from the pig population in Step 3 after outlier removal as the discovery group. Based on the discovery group, perform genome-wide association analysis on the age-to-100 kg body weight phenotype of the pigs obtained in Step 3 after outlier removal to screen for functional loci associated with the age-to-100 kg body weight of pigs.

[0035] Step 5: Screen for genomic backbone sites to capture other genomic effects besides major sites;

[0036] Step 6: Combine the functional loci related to age at which pigs reach 100 kg body weight obtained in Step 4 with the genomic backbone loci obtained in Step 5 to obtain a set of 16,932 markers specific to age at which pigs reach 100 kg body weight, and generate the final 17K SNP sequencing genotyping chip for breeding traits at age at which pigs reach 100 kg body weight.

[0037] Based on the above scheme: the site filtering parameters for identifying polymorphic sites in step 2 are: estimated minimum allele frequency (EAF) > 0.01, sequencing depth ≥ 1.5 IQR; the filtering parameters for genotyping polymorphic sites in step 2 are: minimum allele frequency (MAF) > 0.01, and filling information score (INFO SCORE) > 0.4.

[0038] Based on the above scheme: the screening criterion for screening functional loci related to the age-related traits of pigs reaching 100 kg body weight in step 4 is P<0.0001.

[0039] Based on the above scheme: the specific steps for screening genomic backbone sites in step 5 are as follows: perform LD filtering on the whole-genome SNPs obtained in step 2, with the filtering conditions being: using a window length of 1000bp, the LD ratio of each pair of SNP sites within the window is [missing information]. 2 When the value is greater than 0.25, one of the sites is deleted, and the final genome backbone site is obtained.

[0040] Another objective of this invention is to provide an application of a 17K SNP sequencing and genotyping chip for breeding pigs at 100 kg body weight and age.

[0041] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0042] An application of a 17K SNP sequencing and genotyping chip for breeding pigs at 100 kg body weight and age, characterized by the following steps:

[0043] Step 1: Establish a reference population. Use the sequencing genotyping chip to obtain the genotype data of each individual in the reference population, and record the phenotypic data of the reference population. The effect value of each SNP or different chromosome segments can be estimated through a suitable statistical model.

[0044] Step 2: Then, for each individual in the candidate population, genotyping is performed using the sequencing genotyping chip, and the estimated SNP effect value obtained in the reference population is used to calculate the estimated genomic breeding value of each individual in the candidate population.

[0045] Step 3: Select and retain outstanding individuals based on their breeding value ranking.

[0046] The 17K SNP sequencing and genotyping chip for breeding pigs at 100 kg body weight and age, as described in this invention, has the following beneficial effects:

[0047] 1. The porcine whole-genome sequencing and genotyping chip of this invention does not rely on the complex experimental procedures of traditional SNP chips, such as probe hybridization and targeted sequencing. Instead, it identifies polymorphic sites and performs genotyping through low-depth sequencing technology. This not only reduces the cost of chip design but also significantly reduces the cost of applying the chip to genome selection breeding, achieving a genotyping accuracy of over 98%. This technology is low-cost, highly accurate, convenient, and fast, and can be quickly promoted to genome selection breeding practices.

[0048] 2. The pig whole genome sequencing and genotyping chip of the present invention significantly increases the accuracy of genomic breeding of pigs at 100 kg body weight while reducing the number of markers in traditional SNP chips. This will greatly accelerate the genetic progress of genomic selection of pigs at 100 kg body weight, bringing significant economic benefits and is of great value in pig molecular breeding. Attached Figure Description

[0049] The present invention includes the following figures:

[0050] Figure 1 The distribution map of age-related loci in pigs at 100 kg body weight involved in this invention on the genome. Detailed Implementation

[0051] The present invention will now be described in detail with reference to embodiments and accompanying tables. It should be understood that the following embodiments are given for illustrative purposes only, and the specification and accompanying tables are only for clearly describing one embodiment and are not intended to limit the scope of the invention. The features, operations, or characteristics described in the specification can be combined in suitable ways to form various implementations. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0052] Example 1: A method for fabricating a 17K SNP sequencing and genotyping chip for breeding pigs at 100 kg body weight and age.

[0053] Genomic DNA was extracted from ear tissue samples of 3549 Duroc pigs, and genomic libraries were constructed. Whole-genome resequencing was performed on the genomic libraries of each sample at a rate of 0.5×. Polymorphic sites in the sample population were identified using BaseVar software with the following filtering parameters: estimated minimum allele frequency (EAF) > 0.01 and sequencing depth ≥ 1.5 IQR. Genotyping of all polymorphic sites was then performed using STITCH software with the following filtering parameters: minimum allele frequency (MAF) > 0.01 and infill information score (INFO SCORE) > 0.4, resulting in 11,786,827 SNP sites.

[0054] Record the phenotype, weigh the animals in the range of 85-130 kg, and correct for the age-on-100 kg weight phenotype using the following formula, where the value A depends on the breed and sex.

[0055] Target weight age = Actual age + (Target weight - Actual weight) × (Actual age - A) / Actual weight

[0056] Outliers were removed. A random sample of 1000 pigs was selected as the locus discovery population. Genome-wide association analysis (GWAI) was performed on the age-related phenotypes of pigs reaching 100 kg body weight using a GCTA mixed linear model, with covariates including year, season, and birth weight. Finally, 106 important functional loci were selected based on a p-value < 0.0001.

[0057] Genomic backbone sites are screened to capture other genomic effects besides major effect sites. Low-density (LD) filtering is performed on whole-genome SNPs obtained from low-depth whole-genome sequencing. The filtering conditions are: a window length of 1000 bp, and the LD values ​​of pairwise SNPs within the window are [value missing]. 2 When the value is greater than 0.25, one of the sites is deleted, resulting in a total of 16,831 genomic backbone sites.

[0058] By merging functional loci and genomic backbone loci of pigs at 100 kg body weight day-old, a set of 16,932 markers specific to pigs at 100 kg body weight day-old was obtained, generating the final 17K SNP sequencing genotyping chip for breeding traits of pigs at 100 kg body weight day-old.

[0059] Example 2: Application of 17K SNP sequencing and genotyping chips in genomic selection breeding of pigs reaching 100 kg body weight at age 100 days

[0060] To demonstrate the effectiveness of the site set obtained by this invention, two commercial SNP chips were used as controls: the SMIC No. 1 pig commercial chip and the Neogene pig 80K commercial chip.

[0061] The 3549 pigs in Example 1 were divided into two groups: 1000 individuals from Example 1 served as the discovery group, and the remaining 2549 samples served as the validation group. This avoided information duplication and ensured the robustness of the results. A 5× cross-validation method was used to evaluate the performance of the pig age-specific marker set at 100 kg body weight compared to two commercially available chips. The results showed that the 17K SNP sequencing genotyping chip for pig age-specific traits at 100 kg body weight, developed in this invention, had higher predictive accuracy for these traits, improving by 21.5% compared to the Zhongxin-1 chip and by 18.9% compared to the Newgene pig 80K chip, demonstrating the effectiveness of the locus set in this invention.

[0062] Table 1. Comparison of accuracy between 17K SNP sequencing genotyping chips and traditional chips for phenotypic breeding of pigs at 100 kg body weight.

[0063] site set SMIC No. 1 chip Neogene Pig 80K Chip 17K SNP sequencing and genotyping chip for breeding of pigs reaching 100 kg in weight and at a specific age Accuracy of predicting traits at age when pigs reach 100 kg in weight 0.228±0.03 0.233±0.03 0.277±0.03 ;

[0064] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. The application of SNP locus sets in breeding pigs at 100 kg body weight per day, characterized by: The SNP locus set consists of 16,932 SNP loci, as shown in Table A of the specification.

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