Alternative methodologies for genotyping polymorphisms in the CAST and CAPN1 genes in beef cattle

ABSTRACT The objectives of this study were to genotype single nucleotide polymorphisms (SNP) AF159246:g.2959A>G (CAST/DdeI) and AF248054.2:g.6545C>T (CAPN4751) in beef cattle by PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism), using the restriction enzyme DdeI for both SNP, and describe the use of these genotyping methodologies for the first time. For the SNP located in the CAST gene, new primers were designed, and for the SNP of the CAPN1 gene, the same primers previously described in the literature were used. Bonsmara, Caracu, Senepol, Nelore, and Angus bulls were chosen from among the most used bulls in breeding programs according to their genealogy and the lowest possible degree of parentage between them to ensure an experimental sample representative of the genetic variability in each breed. For the CAST and CAPN1 genes, respectively, the following number of animals were analyzed: Bonsmara (n = 25/22), Caracu (n = 25/26), Senepol (n = 25/24), Nelore (n = 26/26), and Angus (n = 25/24). The accuracy of these methodologies was confirmed by direct sequencing of PCR products generated for the two polymorphisms. The new primers developed for CAST/DdeI SNP detection and the use of DdeI enzyme for CAPN4751 SNP detection were effective in genotyping, since no inconclusive genotypes were observed for these genes. Thus, the genotyping of beef cattle using the PCR-RFLP technique for CAST and CAPN1 genes is robust, relatively inexpensive, and easy to perform in any basic molecular biology laboratory. If the association of these markers with traits of economic interest in beef cattle is confirmed in new studies, these methodologies may contribute to the selection of animals with superior genetics, i.e., with the potential to produce better-quality meat, either by marker-assisted selection or by the inclusion of these polymorphisms in high-density marker panels