A total of 96 individuals were sampled from four populations of Vietnamese local chickens (28 Ho, 32 Dong Tao, 18 Mong, and 18 Mia) distributed in four provinces (Bac Ninh, Hung Yen, Ha Nam, and Hanoi, respectively) in the North of Vietnam. All individuals were from the same conservation program in Vietnam, originating from either private farms or public institutions involved in this program. Neither pedigree nor phenotypic information was available. We used stratified sampling for flocks, ensuring a distance of at least 500 m between the selected flocks and random sampling of one or two individuals in each selected flock. Genomic DNA was extracted from the blood samples of all chickens. The HD array for chickens allows for the simultaneous genotyping of approximately 580,000 SNPs. This array comprises SNPs on 28 autosomal chromosomes, two sex chromosomes (Z and W for poultry), and two linkage groups (LGE64 and LGE 22C19W28). Only SNPs on the 28 autosomes were considered in the present study. The SNPs with more than 10% missing genotypes or minor allele frequencies lower than 1% were removed, yielding a consensus panel of 454,297 autosomal SNPs for the analysis of population genetic structure.

We applied a second quality control within each breed. Specifically, SNPs with within-breed minor allelic frequencies lower than 0.05 or SNPs with significant (P < 0.0001) deviation from the Hardy-Weinberg equilibrium were filtered out. This second filter led to 368,652, 383,792, 405,535, and 432,631 SNPs for Ho, Dong Tao, Mong, and Mia breeds, respectively. Of note, removing monomorphic SNPs within breeds may lead to the elimination of SNPs that are polymorphic across breeds. Due to the large number of available SNPs, we did not consider the potential effect of these filtered markers as important for subsequent analyses. Genetic diversity analyses were performed on the remaining 308,307 SNPs shared by all four breeds.

All statistical analyses were performed using R.