10k liquid-phase chip for pigs based on multiple single nucleotide polymorphisms

A method for SNP marker selection and probe preparation for a 10K liquid phase chip used for multiple single nucleotide-polymorphism, which utilizes whole-genome sequencing data of pig breeds to mine and screen target SNP loci, then designs and optimizes probes for the target SNP loci, ultimately resulting in the determination of the probes;

• wherein the method includes the following steps:
• Step 1:
• determining target SNPs:
• target SNPs are selected as determined target SNPs using the following method:
• (1) preliminary screening of target SNP markers:
• based on whole-genome sequencing data from Duroc, Large White, and Landrace pig breeds, aligned to the pig 11.1 reference genome;
• markers are selected for targeted capture sites, i.e., target SNPs, based on marker spacing and gene function annotations;
• the principles for selecting target SNPs are:
• (a) uniform distribution across chromosomes, with denser distribution at both ends of chromosomes;
• (b) marker spacing greater than 280 Kb;
• (c) important candidate loci for growth, reproduction, feed efficiency, meat quality, and body size traits;
• (d) SNP markers preferably located in QTL regions related to economic traits, as determined by comparison with the QTLdb database;
• markers obtained in this manner are used as target SNPs;
• (2) supplementing and optimizing target SNPs:
• based on the target SNPs obtained in the preliminary screening step, supplement and optimize markers with high polymorphism and moderate regional linkage disequilibrium;
• wherein the principles are as follows:
• (a) polymorphism primarily considers MAF >0.35 in Duroc, Landrace, and Large White pigs;
• (b) the average linkage disequilibrium (r2) with adjacent SNP markers is below 0.85;
• Step 2, designing probes based on the determined target site SNPs:
• utilize the multiple single nucleotide polymorphism technologies to design 1-4 probes, each 110 bp in length, centered on each target SNP;
• each probe covers the target SNP, with a total probe coverage of 165 bp around the target SNP;
• wherein the principles for probe design are:
• 1) select probes with a content between 30% and 80%;
• 2) choose regions with a number of homologous areas ≤5;
• 3) select probe areas that do not contain SSR, N regions;
• and
• Step 3, selecting and optimizing probes containing high-quality mSNPs:
• probes are hybridized and sequenced, and the genotyping quality of mSNPs, including target sites, is detected;
• set a missing rate of NA<0.1, a minimum allele frequency (MAF) ≥0.05, and heterozygosity (Het)<0.5 as standards to screen mSNPs, removing those that do not meet the standards;
• if the probe does not meet the genotype quality control requirements of mSNPs, delete the probe and the corresponding target site SNP;
• the mSNPs that meet the quality inspection requirements are finally used as the target sites of the 10K mSNP liquid-phase chip.