Gene variations of FABP4, SCD, GH, and IGF1R and their associations with meat and carcass traits in sheep : A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

The growing world population is expected to reach nine billion by 2050 (UN, 2019) which positions New Zealand (NZ) as a vital contributor to global food sustainability, particularly in the meat industry. The NZ sheep meat industry is economically significant and continually strives to produce highly desirable and marketable meat as a major protein source for many nations. The production and processing of lamb directly affect its eating quality, which is a key aspect of the value proposition for lamb products offered to consumers. Enhancing economically valuable traits in animal production is a primary focus, achievable through biotechnological approaches such as marker-assisted selection. This method leverages genetic variations within candidate genes that influence production traits, guiding animal breeding efforts to enhance desired characteristics. Improvements in phenotypic traits, including meat quality, growth rate, and carcass composition, provide substantial benefits to the animal production industry. Given that these traits are governed by complex gene interactions, research aimed at identifying variations in candidate genes that drive desirable production traits is increasingly important. In this context, the main objective of this research is to determine the association of the genes for fatty acid binding protein 4 (FABP4), stearoyl CoA desaturase (SCD), growth hormone (GH) and insulin-like growth factor 1 receptor (IGF1R) with meat characteristics such as intramuscular fat (IMF) levels, fat percentage, fatty acid profile, and carcass traits. This understanding aims to facilitate the reliable production of juicy, flavourful, and healthier lamb while maintaining the advantages of the NZ quality lamb production system. A total of 593 NZ lambs were randomly genotyped to identify specific banding patterns representing different variant sequences of each candidate gene. Four genes were initially selected based on their previously demonstrated potential as gene markers for meat and carcass traits. For meat traits, the focus was on FABP4 and SCD, while for carcass traits, GH and IGF1R were examined. The optimised methods for PCR and SSCP were established to balance efficiency and efficacy. Higher PCR annealing temperatures improved band specificity for FABP4 and SCD genotypes. Polyacrylamide gels with 10 - 14% concentration were recommended for SSCP electrophoresis. The new method for SCD reduced processing time by 8 hours, suggesting better time efficiency in genotyping. Overall, of 15 variants were identified: six each from FABP4 and GH, and three from SCD; these three genes were proceeded with nucleotide sequencing analysis and association studies in Chapters 4, 5 and 6. Chapter 4 aimed to identify specific nucleotide sequence variations in FABP4 exon 3 to intron 3 using another set of sheep (N = 422) with carcass data, and meat data were available from 229 of them. Carcass data were obtained from VIAScan analysis produced at the abattoir, and the backstrap muscle (Longissimus dorsi, LD) samples were collected for meat analysis. The collected meat was weighed frozen, freeze-dried and then were analysed for meat characteristics, including dry matter (DM), IMF %, fat %, and the FA profiles. The FAs were individually measured and categorised based on their characteristics (e.g. saturated FA vs. unsaturated FA) and statistically analysed using generalised linear mixed models (GLMM) for association studies linked with gene variants. The breeds involved included Romney, Perendale and Texel x Suffolk-crossed sheep; blood samples from these lambs were genotyped and sequenced using Sanger sequencing. Sequence alignments, translations and comparisons revealed three variants - A, B and C, with six genotypes: AA, AB, AC, BC, BB and CC. Only intronic variations were identified, and one significant association (p < 0.05) was found between variant C and DM %. No associations were observed for other meat or carcass traits. Variant C revealed an association with lower DM %, suggesting that meat with this variant may attribute to juicier meat. Fat % had the strongest correlation with IMF % (r2 = 0.994; p < 0.05) while IMF % was negatively correlated with omega 3 and omega 6 fatty acids. When comparing correlations between meat and carcass traits, fat % and IMF % exhibit moderate relationships with hot carcass weight (HCW) (r² = 0.361; p < 0.05 and r² = 0.398; p < 0.05 respectively). The weak negative correlation between HCW and leg yield suggests that selecting for overall carcass weight may not necessarily improve leg yield – a cut of significant economic importance. Chapter 5 describes the identification of variations in exon 3 of SCD, genotyped from NZ and Nigerian sheep. A total of 230 sheep from both continents were genotyped to analyse sequence variations. Three unique variants were found, named as variants A, B and C with variant frequencies of 98.9%, 0.9% and 0.2% respectively. Variants B and C are reported here for the first time in the literature. Only three genotypes were found: AA, AB and AC with B and C always appearing heterozygous with variant A. No homozygous individuals were found for variants B or C, and genotypes AB and AC were rare at frequencies of 1.7% and 0.4% respectively. Interestingly, variant B was observed across Oceania and Africa; it was found in one sheep breed from NZ (White Dorper) and three sheep from Nigeria (Uda). Due to the rarity, further association studies are recommended with larger populations encompassing various breeds across broader geographical regions. In Chapter 6, GH spanning from exon 2 to partial intron 3 was investigated for gene variation, frequency and its associations with meat and carcass traits were analysed using GLMM analysis on the same sheep group studied in Chapter 4. Genotyping for GH variants utilised the PCR-SSCP method optimised in Chapter 3. Variant A had the highest frequency (77%), followed by variant C (14%), and variant B (9%). Genotype AA was the most abundant (59%), followed by AC (21%) with genotype BB at only 0.8%. Nine polymorphisms were identified across all three variants A, B and C. Variant A contained two single nucleotide polymorphisms (SNPs) in intronic site along with one synonymous SNP in the coding region (c.255A>G p.Pro85Pro), while variant C contained five SNPs relative to the reference sequence and all were located in the non-coding region (intron 2). Variant B had a similar sequence to the reference except at c.174+113G>A. The presence of variant A of ovine GH was associated with an increase in DM % (p = 0.055) and total UFA (p < 0.05) while it was associated with a decrease in MCFAs (p < 0.05). The presence of variant C was associated with increased long chain fatty acids (p < 0.05) and total saturated fatty acid (p < 0.05), and its absence suggested a tendency towards increased HCW (0.05 > p ≤ 0.10). While HCW is a valuable indicator of overall carcass yield, it may not always reflect meat quality due to its dependence on IMF distribution within specific cuts, such as the leg. This underscores the need for comprehensive evaluation of both carcass and meat trait characteristics to fully understand their interrelationships and potential impacts on consumer preferences and market value. This research enhances understanding of polymorphism within FABP4, SCD and GH while assessing their potential as genetic markers for improving sheep meat production. The sequence variations found in this study could potentially be used in marker-assisted selection (MAS) for improving meat and carcass traits in sheep, and the outcomes of this research may contribute valuable insights into sheep genetics and meat production.