Development and application of a 5K targeted liquid SNP panel for Hetian chickens based on genotyping by targeted sequencing.

The Hetian chicken is a nationally protected poultry genetic resource in China, possessing significant conservation value and high potential for commercial development. To facilitate the conservation and sustainable utilization of this valuable indigenous breed, we developed the first breed-specific 5K targeted liquid SNP panel for Hetian chickens using genotyping by targeted sequencing (GBTS) technology. Initially, whole-genome resequencing of 50 Hetian chickens yielded 16,433,661 single nucleotide polymorphisms (SNPs). Following strict quality control and filtering criteria-heterozygosity rate ≤ 50%, missing rate ≤ 10%, minor allele frequency (MAF) > 0.35, and a uniform physical distribution across the genome-5,781 core SNPs were retained to construct the conservation-specific panel. The performance of this panel was subsequently validated using 307 Hetian chicken samples. The results demonstrated a high average call rate of 99.9% and excellent genotyping performance. Furthermore, the panel exhibited robust capabilities for accurate clustering and kinship analyses. The kinship analysis findings further confirmed that this breed-specific panel holds substantial practical value for germplasm resource conservation and the implementation of molecular breeding strategies. Overall, this study highlights the technical advantages of a low-density, cost-effective, and breed-specific liquid SNP panel for precise genotyping, providing a crucial molecular tool to support the protection, genetic mining, and sustainable utilization of Hetian chicken resources.