With the development of high‑throughput sequencing, RNA sequencing has been widely
      used in the identification of candidate genes for complex traits in livestock, and the functional genes
      and mutations with large genetic effects on milk production traits can provide molecular informa‑
      tion for marker‑assisted selection to increase the selection accuracy and accelerate genetic gain in
      dairy cattle. Our previous study on the liver transcriptome of Holstein cows found that acyl‑CoA
      dehydrogenase (ACADVL) and interferon regulatory factor 6 (IRF6) are differentially expressed be‑
      tween dry and peak lactation periods, as well as that they are involved in lipid metabolism and the
      proliferation and differentiation of mammary epithelial cells. Thus, the two genes were considered
      candidates for milk traits. Hence, this study further collected 1186 Holstein cows from 110 sire fami‑
      lies to investigate their genetic associations with milk yield and composition traits. By resequencing
      the entire exons and 2000 bp of the 5′ and 3′ flanking regions of the two genes, we identified eight
      SNPs in ACADVL and eight SNPs in IRF6. Subsequent single‑locus association analyses showed that
      the eight SNPs in ACADVL were all significantly associated with milk fat yield, fat percentage, and
      protein yield (p values ≤ 0.0001–0.0414), and the eight SNPs in IRF6 were associated with milk, fat,
      and protein yields in the first or second lactation (p values ≤ 0.0001–0.0467). Using Haploview 4.2,
      one haplotype block with eight of the SNPs in ACADVL (D’ = 0.99–1.00) and two haplotype blocks in
      IRF6 with three of the SNPs in each were observed (D’ = 0.98–1.00). Similarly, the haplotype combina‑
      tions of ACADVL were significantly associated with milk yield, fat percentage, fat yield, and protein
      yield in the two lactations (p values ≤ 0.0001–0.0125), and those of IRF6 were associated with five
      milk traits (p values ≤ 0.0001–0.0263). Furthermore, with the JASPAR software, it was predicted that
      the SNPs 19:g.26933503T>C in ACADVL and 16:g.73501985G>A in IRF6 changed the transcription
      factor binding sites of ZEB1, PLAGL2, and RHOXF1, implying their impacts on the expressions of
      the corresponding genes. Our findings demonstrated that the ACADVL and IRF6 genes have signif‑
      icant genetic effects on milk yield and composition traits, and the valuable SNPs might be used as
      genetic markers for genomic selection programs in dairy cattle.