Multiomics analysis reveals an association of ASIP with the gray coat color pattern in donkeys

Coat color variation in domestic animals provides important insights into the genetic mechanisms of phenotypic diversity. The gray coat of Hetian gray donkeys is a distinctive breed characteristic, yet its genetic basis remains elusive. We integrated multiomics data from 175 domestic donkeys, including whole-genome sequencing, RNA-seq (n = 6), and Oxford Nanopore Technologies (ONT) sequencing (n = 8). Genome-wide association study (GWAS) and selection signature analyses identified the agouti signaling protein (ASIP) locus as the top candidate associated with the gray coat phenotype. Transcriptomic analysis revealed a 6.17-fold increase in ASIP expression in Hetian gray donkeys, suggesting that elevated ASIP levels may inhibit eumelanin synthesis. Further, comparative ONT analysis identified a 30 bp deletion in the second intron of ASIP. Dual-luciferase assays demonstrated that this deletion significantly enhances transcriptional activity. The increased ASIP expression appears to prevent melanocyte maturation, resulting in hypopigmentation in the skin and hair of Hetian gray donkeys. Our study provides functional evidence that this structural variant in ASIP modulates pigmentation by enhancing its expression, thereby offering mechanistic insight into both a distinctive donkey breed phenotype and the evolutionary processes shaping coat color diversity in equids.