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Background  5,10-Methylenetetrahydrofolate reductase (MTHFR) plays a central role in folate metabolism by irreversibly converting 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate, a predominant circulating form of folate. Folate is reportedly important for milk protein synthesis, and MTHFR may be a key regulatory point of folate metabolism for milk protein synthesis in mammary epithelial cells. Prior to this study, polymorphisms of the  MTHFR  gene were not associated with milk production traits from a breeding perspective. Single nucleotide polymorphisms (SNPs) at microRNA (miRNA) binding sites (miR-SNPs) can affect gene expression. This study aimed to identify the effects of miR-SNPs (g.2244A>G and g.2264A>G) in the caprine  MTHFR  3' UTR on the milk production traits of dairy goats.    Results  Guanzhong dairy (GD,  n  = 325) goats were used to detect SNPs in the caprine  MTHFR  3' UTR by DNA sequencing. Two novel SNPs (g.2244A>G and g.2264A>G) were identified in the said region. The homozygous haplotype A-G of the SNPs g.2244A>G and g.2264A>G was significantly associated with milk yield and milk protein levels in GD goats ( P  < 0.05). Functional assays indicated that the  MTHFR  2244 A → G substitution could increase the binding activity of hsa-miR-1266 with the  MTHFR  3' UTR. The  MTHFR  2264 A → G substitution could decrease the binding activity of hsa-miR-616 with the  MTHFR  3' UTR. In addition, we observed a significant increase in the  MTHFR  mRNA levels of homozygous haplotype A-G carriers relative to those of homozygous haplotype G-A carriers. These results indicated that both SNPs altered the  MTHFR  mRNA levels. These altered levels of  MTHFR  mRNA may account for the association of SNPs with milk production traits.    Conclusions  This study is the first to report that the g.2244A>G and g.2264A>G polymorphisms were associated with milk production traits in GD goats. Further investigations should explore the underlying miRNA-mediated mechanisms that are modified by the g.2244A>G and g.2264A>G SNPs. The current study evaluated these SNPs as potential genetic markers in goats, with potential applications in breeding programs.

Two Mutations in the Caprine MTHFR 3'UTR Regulated by MicroRNAs Are Associated with Milk Production Traits