Genome-wide scan and fine-mapping nominate ANAPC4 as a candidate modulator of Chinese Holstein heifer puberty timing

Dairy fertility strongly affects farm sustainability because young cows must be raised for many months before they begin producing milk. 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 whole-genome sequencing and genotype imputation, a total of 7,964,217 SNPs were retained for genome-wide association analysis (GWAS) and window-based variance partitioning. Among the seven traits evaluated—AFS, age at first calving (AFC), first-to-last insemination interval in heifers (IFLh), number of services per conception in heifers (NSh), conception rate in heifers (CRh), birth weight of heifers (BiWh), and gestation length in heifers (GLh)—AFS exhibited the highest heritability, with a moderate estimate of h²=0.276. Three candidate regions prioritized by significance and additive variance jointly explained 0.91% of the genetic variance. On BTA6, the association peak spans ZCCHC4 and ANAPC4; notably, ANAPC4 contributes to estrogen-induced oocyte maturation. In blood (n=648), cis-expression quantitative trait loci (cis-eQTL) mapping and co-localization analysis provided strong evidence that the GWAS locus colocalizes with ANAPC4 expression, with the posterior probability of shared causality (PP4) reaching 0.921. SuSiE fine-mapping pinpointed the association to a ~30 kb region comprising a 20-variant credible set that segregates into two high linkage disequilibrium (LD) haplotypes (H0 and H1), with the H1H1 genotype linked to elevated ANAPC4 expression and a ~2.1-day reduction in AFS. 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.