Validation of stearoyl-coA desaturase gene TaqMan probe-based SNP for genotyping Tattykeel Australian White MARGRA lamb for health-beneficial omega-3 long-chain fatty acids and intramuscular fat content

The Tattykeel Australian White (TAW) branded MARGRA lamb is a premium Australian sheep breed developed through decades of careful linebreeding, with the aim of improving the natural levels of health beneficial omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), micro-marbled intramuscular fat (IMF) content, and low fat melting point (FMP) in the highly priced Longissimus dorsi muscle. These traits are key attributes contributing to the well documented superior meat eating quality of the TAW lamb, hence, fundamental to the TAW breeding program. The focus on Stearoyl-CoA Desaturase (SCD) gene is relevant because it plays an essential part in the biosynthesis of n-3 LC-PUFA and IMF metabolism. Previous studies had identified and established significant associations between the g.23881050T > C single nucleotide polymorphism (SNP) in the SCD gene and meat eating quality traits. Therefore, the aim of this study was to develop a reliable, fast, cost-effective, and accurate TaqMan probe-based Real-Time PCR assay for genotyping the identified SCD SNP in 118 TAW, Poll Dorset, Martin More sheep, and Angus bulls (as a distinguishing species control). By designing allele-specific TaqMan probes targeting the polymorphic locus, the assay accurately identified two genotypes, homozygous CC and heterozygous CT, at an estimated cost of AUD 2.16 and processing time of 49.30 minutes per 96-well sample plate. The genotype frequencies for CC and CT were 0.58 and 0.42 in rams, and 0.57 and 0.43 in ewes, respectively. The major and minor allele frequencies in TAW rams and ewes were 0.21 and 0.79, while in young TAW rams, they were 0.17 and 0.83, respectively. Both Poll Dorset and Martin More sheep exclusively displayed the CC genotype at a minor allele frequency of 1.00. The findings validate the identified SNP g.23881050T > C as a suitable DNA marker for breeding for meat quality traits. and the validated TaqMan probe-based genotyping assay is a simple, reliable, fast, precise and cost-effective tool for genotyping TAW. Therefore, the developed and validated TaqMan probe herein, can facilitate genetic selection within the TAW breeding program, and assist in streamlining efforts to enhance meat eating quality of the breed.