Identification of Polymorphisms of Gene CSN2 of B Casein in Greek Cow Breeds (Holstein) by Restriction Fragment Length Polymorphism

Abstract

The research focused to detect and identify genetic polymorphisms in exon 7 of the β-casein CSN2 gene in blood samples from Greek Holstein cows. For this purpose, DNA was extracted from 120 blood samples of cows. The desired region of exon 7 was amplified by PCR, resulting in a 121 bp product. The PCR product was digested by restriction fragment length polymorphism (RFLP) method. The results suggest that the A1A2 genotype prevails over the others. Specifically, of the 120 cattle, 72 showed triple bands of 121 bp, 86 bp, and 35 bp indicating the A1A2 genotype. The 42 cattle showed a single band at 121 bp, indicating that they carried the A1A1 genotype. The remaining 6 showed only two bands of 86 and 35 bps, indicating that they carried the A2A2 genotype. In the total population of heterozygotes A1A2-0.60 were the most frequent, while homozygotes A2A2-0.06 were the least frequent ones. This suggests a slight superiority of allele A-0.65.

DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein

“…With respect to DNA extraction methods, methods 1 and 2 were a direct applications of a commercially available kits, NucleoSpin Tissue (Usman et al, 2014;Vougiouklaki et al, 2020) and NucleoSpin Blood (Macherey-Nagel, Düren, Germany), both kits are based on the binding of DNA to a silica gel membrane to get high-quality DNA. The main difference between them was the incubation time after adding proteinase K, which is was not necessary for NucleoSpin Blood and it was 2 h for the NucleoSpin Tissue kit.…”

“…Apart from qPCR methods, in Mayer et al (2021) milk proteins were directly phenotyped through isoelectric focusing to discriminate A1 and A2 β-casein protein variants. However, most studies were focused on the genotyping of the CSN2 gene to determine the frequency of these genotypes in different cattle breeds (Masoumeh Firouzamandi et al, 2018;Mayer et al, 2021;Miluchová et al, 2014;Rangel et al, 2017;Vougiouklaki et al, 2020). Identification of animal genotypes may not be enough to A2 milk authentication (Giglioti et al, 2020).…”

Método de genotipagem para detecção de polimorfismos no gene CSN2 em vacas leiteiras Girolando

“…Aproximadamente 80% das proteínas presentes no leite bovino são classificadas como caseínas (α-s1, α-s2, kappa e β-caseína), e 20% são compostas por proteínas séricas (Vougiouklaki et al, 2020). A β-CN (Beta caseína) composta por 209 aminoácidos representa 45% das caseínas presentes no leite bovino.…”

Comparative analysis of A1 and A2 allele detection efficiency for bovine CSN2 gene by AS-PCR methods

“…Sequencing is conducted in some cases, but regardless of its efficiency, at this stage, it is not always reasonable for routine large-scale population studies (Dai et al, 2016). The genotyping of cattle by alleles A 1 and A 2 at the beta-casein locus is conducted in different regions of the world which demonstrates the increasing interest in this problem (Zepeda-Batista et al, 2015;Vougiouklaki et al, 2020;Ivankovi´c et al, 2021). In recent years, the interest in genotyping cattle from different breeds has been noted in Ukraine, which is confirmed by a number of publications (Ladyka et al, 2020;Ladyka et al, 2021).…”