Analysis of acetyl-CoA carboxylase gene polymorphisms in Valle del Belice dairy breeds

Abstract

Acetyl-CoA carboxylase is the flux-determining enzyme in the regulation of fatty acid synthesis within animal tissues. The expression of the mammary gland isoform of this enzyme, during lactation, is regulated by the acetyl-CoA carboxylase-α (ACACA) gene. The ovine ACACA gene, with 51 coding exons, is responsible for phenotypic variability observed in milk fat content and milk fatty acids (FAs) composition. However, before attempting association analyses between this enzyme and phenotypic traits of interest, a study on the genetic variability within this locus is required. The aim of this work was to sequence the encoding region of ACACA gene in Valle del Belice dairy ewes in order to identify polymorphic sites. Individual milk samples from 113 Valle del Belice ewes were collected. After fatty acid methyl esters (FAMEs) analysis of all samples, a selective genotyping approach was used. As criteria, the mean values of each FA±2 standard deviations were used to identify the highest and lowest phenotypic values. Two sets of 8 animals each were genotyped in a SNP discovery study of the ACACA gene. A preliminary analysis was performed with SAS using the PROC ALLELE procedures to estimate allele frequencies and Hardy-Weinberg Equilibrium (HWE) of identified polymorphic sites. The exact position of the SNPs was detected according to NCBI mRNA Reference Sequence (Acc. No NM_001009256). Sequencing analysis and alignment of the obtained sequences showed the presence of 14 polymorphic sites in the ACACA exons. In particular: C1441T in exon 11; 2 SNPs in exon 13 (T1783G and T1834C); T2347C in exon 17; A2575G in exon 19; 2 SNPs in exon 37 (C4534T and G4579A); and C5383T in exon 42. The most polymorphic was exon 53 which showed the presence of 6 SNPs (T6928C, C6991T, C6995T, G7005A, C7082G, and T7095C). All SNPs were in HWE. Analysis of SNPs frequencies showed that major allele was always at frequency >50% in all cases. The characterization of ACACA gene reported herein laid the basis for further association analyses needed to evaluate the potential use of these SNPs as genetic markers for fat content and FAs composition in sheep milk.