A pig teat number trait breeding 130K SNP sequencing typing chip and application

Abstract

This invention relates to a 130K SNP sequencing genotyping chip for breeding porcine nipple number traits and its application. This invention utilizes large-scale low-depth whole-genome resequencing technology to specifically screen for functional markers related to porcine nipple number, significantly eliminating noisy sites in the sequencing data while retaining functional sites related to nipple number. This results in a 130K SNP sequencing genotyping chip for breeding porcine nipple number traits, reducing genotyping costs while greatly improving the accuracy of nipple number trait breeding.

Description

Technical Field

[0001] This invention relates to the fields of genome breeding, functional genomics, and molecular biology, specifically to a 130K SNP sequencing and genotyping chip for breeding the number of pig nipples 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] Reproductive traits in pigs play a crucial role in production efficiency and economic benefits, and have always been a focus of breeding research and production. As an important organ for lactation, the teat is closely related to milk production performance and is most likely to be used as a secondary selection trait for pig reproductive traits. Furthermore, the number of teats in pigs is more strongly constrained by genetics than some other economic traits, and is less susceptible to variation due to feeding and management conditions. At the same time, the number of teats is easy to measure and remains stable after birth. However, the teat number trait is a complex quantitative trait, and it is difficult to directly and effectively apply it to the breeding industry using only a few molecular markers. Therefore, genomic selection technology is still needed to achieve rapid selection of individuals with this trait. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a 130K SNP sequencing genotyping chip for breeding porcine nipple number traits. This invention utilizes large-scale low-depth whole-genome resequencing technology to specifically screen for functional markers related to porcine nipple number, significantly eliminating noisy sites in the sequencing data while retaining functional sites related to nipple number. This results in a 130K SNP sequencing genotyping chip for breeding porcine nipple number traits, reducing genotyping costs while greatly improving the accuracy of nipple number trait breeding.

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

[0007] A 130K SNP sequencing and genotyping chip for breeding pig nipple number trait, characterized by containing 130,134 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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[0140] Another objective of this invention is to provide a method for fabricating a 130K SNP sequencing and genotyping chip for breeding pig nipple number traits.

[0141] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0142] A method for fabricating a 130K SNP sequencing genotyping chip for breeding pig nipple number trait, characterized by comprising the following steps:

[0143] 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;

[0144] 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;

[0145] Step 3: Collect the number of teats phenotypes of individuals in the pig population, using the number of valid teats within one week after birth as the standard, and remove outliers;

[0146] 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 outlier removal phenotype of pig nipples obtained in Step 3 to screen functional loci associated with the number of pig nipples.

[0147] Step 5: Screen for genomic backbone sites to capture other genomic effects besides major sites;

[0148] Step 6: Combine the functional loci related to the number of pig nipples obtained in Step 4 with the genomic backbone loci obtained in Step 5 to obtain a set of 130,134 markers specific to the number of pig nipples trait, and generate the final 17K SNP sequencing genotyping chip for breeding the number of pig nipples trait.

[0149] Based on the above solutions:

[0150] The site filtering parameters for identifying polymorphic sites in step 2 are: estimated minimum allele frequency (EAF) > 0.01, and sequencing depth ≥ 1.5 IQR;

[0151] In step 2, the genotyping of polymorphic sites is performed, and the filtering parameters for the genotyping results are: minimum allele frequency (MAF) > 0.01 and fill information score (INFO SCORE) > 0.4.

[0152] Based on the above solutions:

[0153] The screening criterion for functional loci related to the number of pig nipples in step 4 is P<0.01.

[0154] Based on the above solutions:

[0155] The specific steps for screening genomic backbone sites in step 5 are as follows: LD filtering is performed on the whole-genome SNPs obtained in step 2. The filtering conditions are: a window length of 1000 bp, and the LD values ​​of each pair of SNP sites within the window are... 2 When the value is greater than 0.40, one of the sites is deleted, and the final genome backbone site is obtained.

[0156] Another objective of this invention is to provide an application of a 130K SNP sequencing and genotyping chip for breeding pig nipple number traits.

[0157] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0158] An application of a 130K SNP sequencing genotyping chip for breeding pig nipple number trait, characterized by the following steps:

[0159] 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.

[0160] Step 2: Then, for each individual in the candidate population, genotyping is performed using the sequencing genotyping chip method, 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.

[0161] Step 3: Select and retain outstanding individuals based on their breeding value ranking.

[0162] The beneficial effects of the 130K SNP sequencing and genotyping chip for porcine nipple number trait breeding and its application described in this invention are as follows:

[0163] 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.

[0164] 2. The porcine whole-genome sequencing and genotyping chip of the present invention significantly increases the accuracy of genomic breeding for nipple number traits while reducing the number of markers in traditional SNP chips. This will greatly accelerate the genetic progress of genomic selection for nipple number traits, bring significant economic benefits, and is of great value in porcine molecular breeding. Attached Figure Description

[0165] The present invention includes the following figures:

[0166] Figure 1 The distribution map of nipple number-related loci on the genome involved in this invention. Detailed Implementation

[0167] 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.

[0168] Example 1: A method for fabricating a 130K SNP sequencing and genotyping chip for breeding pig nipple number trait.

[0169] 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.

[0170] Records of the nipple number phenotype were collected, using the number of valid nipples within one week of birth as the criterion, excluding outliers. A randomized population of 1000 pigs was selected from the entire sample as the locus discovery group. Genome-wide association analysis (GWAI) was performed on the nipple number phenotype based on this population using a mixed linear model with covariates including year, season, and birth weight. Finally, 102,677 important functional loci were identified based on a p-value < 0.01.

[0171] Genomic backbone sites are screened to capture genomic effects other than 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 0.40, one of the sites is deleted, resulting in a total of 27,756 genomic backbone sites.

[0172] By merging functional loci and genomic backbone loci for nipple number, a set of 130,134 markers specific to the pig nipple number trait was obtained, which were used to generate the final 130K SNP sequencing genotyping chip for pig nipple number trait breeding.

[0173] Example 2: Application of 130K SNP sequencing and genotyping chip in genomic selection breeding for porcine nipple number trait breeding

[0174] 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.

[0175] 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 nipple number-specific marker set and two commercial chips. The results showed that the 130K SNP sequencing and genotyping chip for pig nipple number trait breeding developed in this invention had higher prediction accuracy for the pig nipple number trait, improving accuracy by 12.5% ​​compared to the Zhongxin-1 chip and by 11.5% compared to the Newgene pig 80K chip, demonstrating the effectiveness of the locus set in this invention.

[0176] Table 1. Comparison of accuracy between 130K SNP sequencing genotyping chip and traditional chip in porcine teat number trait breeding.

[0177] site set SMIC No. 1 chip Newgen Pig 80K Chip 130K SNP sequencing and genotyping chip for breeding of pig nipple number trait Accuracy of predicting nipple count trait 0.352±0.04 0.355±0.04 0.396±0.04 ;

[0178] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. A 130K SNP sequencing and genotyping chip for breeding pig nipple number trait, characterized in that, It contains 130,134 SNP sites, as shown in Table A.

2. An application of the 130K SNP sequencing and genotyping chip for breeding porcine nipple number trait as described in claim 1, characterized in that, Includes the following steps: 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. 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. Step 3: Select and retain outstanding individuals based on their breeding value ranking.

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