PCR-based genotyping of SNP markers in sheep

Abstract

Single nucleotide polymorphisms (SNPs) are the main type of variation in genome, enabling them to be associated with traits of economic importance in livestock. Genome-wide association studies (GWAS) have led to the discovery of SNPs associated with desirable traits in sheep. However, in these studies, SNPs are genotyped by high-throughput methods in genome scale, which are expensive and require sophisticated equipment and analysis methods. Therefore, the goal of this study was to develop a reliable, rapid, and inexpensive polymerase chain reaction (PCR)-based method to genotype a medium number of animals for a few candidate SNPs previously associated with desirable phenotypes in sheep by GWAS, using markers associated with gastrointestinal nematode resistance as a model. DNA extracted from white-blood cells of 150 sheep was submitted to PCR amplification followed by agarose gel electrophoresis and determination of banding pattern. Tetra-primer ARMS-PCR was successfully optimized after changes in annealing temperature; annealing and extension times; concentration of MgCl and DNA; ratios of inner, outer, forward and reverse primer; and addition of adjuvants, for genotyping the OAR2_14765360, OAR6_81718546, OAR11_62887032, and OAR12_69606944 SNPs in sheep. An extensive optimization of tetra-primer ARMS-PCR resulted in a suitable, simple, cost-effective PCR-based method of genotyping four SNP markers previously detected by chip arrays.

Single-Nucleotide Polymorphisms Identified within Exon 2 of Fertility-Associated Bone Morphogenetic Protein (BMP15) Gene in Three Romanian Sheep Breeds

“…Moreover, although relatively cost-efficient, sequencing could be difficult to consider and/or to perform in real farm settings if fast and efficient sheep genotyping is required. Such mutational hotspots could be relevant for various PCR applications, such as the amplification refractory mutation system PCR that was previously employed for identifying SNP markers in sheep [62].…”

Investigating mutations in the genes GDF9 and BMP15 in Pelibuey sheep through the amplification-refractory mutation system with tetra-primers

“…SNP are changes or mutations in a nitrogenous base in the DNA sequence that can be used as molecular markers (Ahlawat et al, 2014). Currently, SNP are used to identify allelic variants and their genotypes (Ahlawat et al, 2014;Niciura et al, 2018). In this study, T-ARMS-PCR allowed the identification of polymorphism G4, and in the genotyping of the SNP, 5% of the sheep presented the mutant genotype and 95% of the wild-type genotype.…”

“…Polymorphism is the main source of variation in the DNA of an organism and can be used as a molecular marker to identify allelic variants associated with economically important characteristics (Niciura et al, 2018). In the recognition of these allelic variants, some methodologies have been used to identify SNP and their genotypes in ewes.…”

“…These methods include DNA sequencing, restriction fragment length polymorphism through Polymerase Chain Reaction (PCR-RFLP), single-strand conformation polymorphism (SSCP), hybridization with TaqMan fluorescence probes, and DNA Microarrays, among others (Ahlawat et al, 2014;Niciura et al, 2018). The disadvantage of these methodologies is the high cost of reagents as well as the requirement for specialized materials and equipment for sample processing (Niciura et al, 2018). In this context, Newton et al (1989) developed an Amplification-Refractory Mutation System (ARMS-PCR).…”

Genome-Wide Association Study for Haemonchus contortus Resistance in Morada Nova Sheep

“…DNA was extracted by saline precipitation [59] from venous blood samples obtained from lambs. DNA integrity was confirmed via 1% agarose gel electrophoresis, and the DNA concentration and purity (260/280 absorbance ratio between 1.8 and 2.0) were estimated using a NanoDrop 2000 spectrophotometer.…”