Investigation of genes involved in meat production in cattle : A thesis submitted in partial fulfilment of the requirement for the Degree of Doctor of Philosophy at Lincoln University

In recent years, there has been both an increase in demand for beef due to the growing human population and an increase in demand for beef with improved eating quality. Advances in breeding technology have allowed the development of various DNA selection tools, such as the use of marker-assisted selection to improve beef quantity and quality. However, this approach relies on the availability of reliable genetic markers for desirable traits. Accordingly, this study aimed to investigate polymorphism in selected genes that potentially regulate selected beef traits. The genes selected for study encode the fatty acid-binding protein 4 (i.e., FABP4), stearoyl CoA desaturase (i.e., SCD) and lipin 1 (i.e., LPIN1), and they were chosen based on the role they appear to play in lipid metabolism and energy regulation. Genomic DNA and data were collected from the Simmental (n = 173) and Charolais (n = 70) heifers born and raised in New Zealand, and from White Fulani (n = 48) cattle from Nigeria. The phenotypic data collected from the New Zealand cattle breeds included live weight and IMF content at approximately 14 to 19 months of age. As these were stud breeding stock, IMF levels were measured for live animals using a real-time ultrasound imaging approach. The genes selected for study were analysed using polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) analyses. This study confirmed that the genes investigated were polymorphic and that variation in these genes was associated with beef traits. Four FABP4 variants (named variants A to D) in the exon 3-intron 3 region were identified with these containing five nucleotide substitutions. Two of these nucleotide substitutions were in exon 3 of FABP4 (c.328G/A and c.348G/C), and the remaining three (c.348+34T/C, c.348+56T/C, and c.348+303T/C) were in intron 3. The substitution c.328G/A is non-synonymous and would result in the replacement of valine with methionine (p.Val110Met). The variation at c.348G/C is located at the end of exon 3 and in a region that may affect transcript splicing. Variants A, B, and C were observed in all the cattle investigated, whereas variant D was exclusively found in the Nigerian White Fulani cattle. Association analysis revealed that the presence of variant A was associated with a 7.5% reduction in IMF levels, whereas no significant association was detected with cattle live weight. The study revealed that the gene for stearoyl CoA desaturase was variable and associated with IMF levels and the live weight of the New Zealand beef cattle. There were nine SNPs identified overall in the two SCD regions amplified. Three of SCD SNPs were in exon 5 (c.702A/G, c.762T/C and c.878T/C) and one in intron 5 (c.880 + 105A/G). The nucleotide substitution at c.878T/C would result in an amino acid change from valine to alanine at the 293rd residue (p.Val293Ala). Five nucleotide variants were identified in the 3’-untranslated region (3’UTR) analysed (c.*1783A/G, c.*1883C/T, c.*1884G/A, c.*1984A/G, and c.*2066T/C/G), giving a total of five unique variant sequences (A, B, C, D, and E). Association analysis revealed that the nucleotide sequence variation in SCD that would result in p.293Ala was associated with both increased IMF levels and increased live weight for the beef cattle studied. In the 3’UTR of SCD, variant C was associated with lower IMF levels and live weights. The SCD variant B, on the other hand, was found to only be associated with reduced live weight, and not IMF levels in cattle studied. Bovine LPIN1 was polymorphic, with six SNPs found across the two regions of the gene studied. Region 1 consists of the transcriptional activating domain, while region 2 spanned the DXDXT motif. Five sequences (named variants A – E) and five nonsynonymous SNPs were observed in region 1. In region 1, the presence of LPIN1 variant (haplotype) E was associated with a 10 kg (2.2%) increase in live weight. The SNP genotype AG at c.1527 was associated with a significant (P = 0.023) increase of 11 kg in live weight compared to SNP genotype AA. This c.1527G/A SNP would result in the presence and absence of arginine amino at position 385 of lipin 1. The c.1527A/G SNP was not associated with variation in intramuscular fat (IMF) levels (P = 0.233). The SNP c.1584T/C was also associated with variation in live weight, where genotype TC was found to be associated with an 11 kg increase in weight. Like c.1527A/G, SNP c.1584T/C only appeared to influence live weight (P = 0.017) and not IMF level (P = 0.227). The TC and TT SNP genotypes would underpin the presence and absence of methionine at amino acid 404 of lipin 1 (p.M404T), with the presence of threonine being associated with increased live weight. The impact of c.1527A/G was like that of c.1584T/C because both SNPs were tightly linked. In conclusion, FABP4, SCD and LPIN1 genes are polymorphic and contain potential genetic markers associated with IMF level and live weight in beef cattle in New Zealand.