Additional_file_1.xls

Age at first service (AFS) is a key driver of reproductive efficiency and rearing costs in dairy systems. We analyzed Chinese Holstein heifers to dissect the genetic architecture of AFS and nominate functional candidates. After low-coverage WGS and imputation, 7,964,217 SNPs (8,574 heifers with phenotypes) were used for genome-wide association and window-based variance partitioning; AFS showed moderate, top-ranking heritability among fertility traits (h² = 0.276). Three candidate regions prioritized by significance and additive variance jointly explained 0.91% of the genetic variance. The peak signal on BTA6 spans ZCCHC4/ANAPC4. In blood (n = 648), tensorQTL cis-eQTL mapping followed by COLOC showed strong colocalization of the GWAS peak with ANAPC4 expression (PP.H4 = 0.921). SuSiE fine-mapping concentrated association into a ~30-kb interval comprising a 20-variant credible set that forms two high-LD haplotypes (H0/H1); H1H1 was associated with higher ANAPC4 expression and a shorter AFS (~1.2 days). Motif analyses suggested allele-specific TF-binding differences between haplotypes consistent with regulatory effects. Taken together, these findings pinpoint a fine-mapped regulatory haplotype that upregulates ANAPC4 expression and is associated with earlier AFS, providing actionable markers for genomic selection to advance heifer puberty timing and improve farm profitability.