Identification of alternative splicing events in cows with A1A1 and A2A2 β-casein genotypes using large gap read mapping.

backgroundThe A2 β-casein milk variant has gained interest due to potential health benefits, which has contributed to the rise of A2A2 genotypes within dairy herds. Although the A1 to A2 amino acid substitution in β-casein does not directly regulate gene expression, selective breeding for the A2A2 genotype may have indirect transcriptomic consequences in the mammary gland.

resultsIn this study, we investigated whether the A1A1 and A2A2 β-casein genotypes are associated with differential mRNA isoform expression in bovine milk fat globules (MFG) using RNA-Sequencing technology (RNA-Seq). A total of 619 million paired-end reads from 14 MFG cDNA libraries were analysed. In total, 3,454 DE mRNA isoforms were identified (FDR < 0.05, |FC|> 2), of which 823 were mRNA isoforms with annotated associated genes, 2,067 were mRNA isoforms with annotated associated genes but novel transcript lengths and 586 were novel mRNA isoforms with non-annotated associated genes. Functional analysis of the 2,954 DE mRNA isoforms with annotated gene names revealed 523 enriched metabolic pathways (p < 0.05). Among these pathways, patterns consistent with mitochondrial dysfunction and reduced mitochondrial fatty acid β-oxidation were observed in A2A2 cows, together with indications of reduced mitochondrial biogenesis and altered mitochondrial RNA degradation, suggesting adjustments in cellular energy metabolism. Additionally, differences in isoforms related to ER-Golgi communication, vesicle trafficking and protein folding pointed to subtle modifications in secretory pathway dynamics. Regarding lipid metabolism, A2A2 cows showed signs of increased fatty acid uptake but reduced capacity for de novo synthesis of fatty acids, triglyceride and cholesterol synthesis, as well as for lipid droplet formation.

conclusionsThese findings suggest that the A2A2 genotype would be associated with isoform level changes likely driven by alternative splicing events. Further functional validation will be necessary to assess the biological relevance of the observed differences and their potential implications for genetic selection for the A2 β-casein variant.