Comprehensive multi-omics analyses reveal small intestinal genetic mechanisms regulating milk protein trait in yak

ABSTRACT Yak milk is recognized for its superior nutritional quality, yet the biological basis underlying its high milk protein content remains poorly understood, particularly regulatory mechanism of small intestine. This study compared the major nutrients and amino acid composition of raw milk among yaks, cattle-yaks and cows, revealing the characteristics of high protein and rich amino acids in yak milk. We annotated 16 major cell types from a total of 27,026 cells by constructing the first single-cell transcriptome atlas of yak small intestine. Among them, intestinal stem cells and tuft cells exhibited strong proliferation and differentiation potential, contributing to the maintenance of efficient protein absorption. Our multi-strategy GWAS framework by integrating genomics and scRNA-seq data further identified candidate genes associated with milk protein content (SCP2, ETV6, ACSS2, and WWOX), which are mainly involved in amino acid utilization and energy regulation. Notably, WWOX was consistently identified across multiple analyses. It may enhance protein absorption efficiency in the small intestine, thereby providing sufficient substrates for milk protein synthesis and ultimately increasing milk protein content in yaks. In summary, these findings demonstrate that functional specialization of the small intestine contributes to enhanced milk protein content in yaks and highlight the importance of integrating phenotypic, cellular, and genetic data to understand complex traits. This work provides a novel perspective on the regulation of milk protein and offers potential targets for genetic improvement of milk quality in yaks and other ruminants.