A novel duplex ACRS-PCR for composite CSN1S1–CSN3 genotype discrimination in domestic buffalo

Abstract

This short communication aimed to develop a fast and straightforward method for the simultaneous discrimination in buffaloes of the alleles CSN1S1 A/B and CSN3 X1/X2 using a single protocol. DNA was isolated from 219 individual blood samples. A duplex artificially created restriction site (ACRS-PCR) was accomplished using two pairs of primers generating 86 bp (CSN1S1) and 160 bp (CSN3). Amplicons were contemporary digested by MboI and HinfI for the identification of genotypes. The double simultaneous amplification and digestion proved to be effective for allele identification. The method resulted particularly quick due to the small PCR amplicons and fast digest enzymes that allowed both a rapid amplification (about 1 h and 40 min) and digestion in 10 min. Population analysis indicated that minor allele frequencies were CSN1S1 A (0.425) and CSN3 X2 (0.306). Linkage disequilibrium (LD) showed r 2 ¼0.46, and haplotype analysis revealed all four possible combinations with higher frequency for the CSN1S1 B-CSN3 X1 (0.553). Considering the tremendous economic impact of the CSN1S1-CSN3 variants on the dairy production in buffalo, this method, applicable immediately after the birth from any DNA source, may speed up the selection of sires and dams' lines with more favourable genotypes. HIGHLIGHTSCSN1S1-CSN3 composite genotype influences dairy performances. Duplex ACRS-PCR identifies the CSN1S1-CSN3 composite genotype in a single protocol. The duplex ACRS-PCR method may speed up the selection of lines with more favourable genotypes.

Sequencing and Characterization of αs2-Casein Gene (CSN1S2) in the Old-World Camels Have Proven Genetic Variations Useful for the Understanding of Species Diversification

“…Only a few SNPs (4 out of 18 in Bactrian and 2 out of 16 in dromedary) were found in the retrotransposons, contributing to 17.6% of the total genetic diversity found. This result reflects a lower level of genetic diversity found in the casein genes of camelids [3,14,[18][19][20]22,23,61,62] compared to ruminants [13,[63][64][65][66]. These findings are consistent with the observations made by other authors, indicating a significant reduction in genetic diversity due to at least two bottlenecks during the history of camel domestication [56,67,68].…”

CSN1S1, CSN3 and LPL: Three Validated Gene Polymorphisms Useful for More Sustainable Dairy Production in the Mediterranean River Buffalo

“…Conversely, the deviation from the HW principle for CSN1S1 (χ 2 = 5.06) was unexpected, given the findings of previous studies [21,22]. However, since 2021, the Italian buffalo population has been under selective pressure for the SNP AJ005430:c.578C>T (https://www.risbufala.it/?page_id=58841, accessed on 6 May 2024).…”

“…Genotyping was accomplished using PCR-based methods. In particular, a duplex Artificially Created Restriction Site (ACRS)-PCR described by Pauciullo et al [22] was performed for the AJ005430: c.578C>T at the CSN1S1 (αs1-CN) and HQ677596: c.536C>T at the CSN3 (k-CN). Additionally, Restriction Fragment Length Polymorphism (RFLP)-PCR, as described by Gu et al [28] and Gu et al [27], was used for genotyping the FM876222: g.133A>C at SCD (Stearoyl-CoA desaturase) and the AWWX01438720.1: g14229A>G at LPL (Lipoprotein lipase), respectively.…”

“…Milk yield [14][15][16][17][18], total protein and caseins [19][20][21][22][23], fat content, fat percentage and fatty acid composition [18,20,[24][25][26][27][28][29][30], milking time [14], etc., are among the most studied traits and are of great interest to breeders' associations due to their direct link to cheese yield and economic profitability. Genetic variability and associations with dairy traits have been found for many genes of economic interest (CSN1S1, CSN1S2, CSN3, SCD, LPL, OXT, OXTR, etc.).…”

Genotyping of Cattle by Allelic Variants A1 and A2 of the Beta-Casein Gene: Employing Different Methodological Approaches (As-PCR and Acrs-Pcr)

“…В основі методу ACRS-PCR (Artificially Created Restriction Site Polymerase Chain Reaction) лежить використання ендонуклеази рестрикції, але, на відміну від класичного PCR-RFLP, сайт для ферменту наявний тільки в ампліконі, а не у вихідному фрагменті гену (Pauciullo et al, 2021). Створення штучного сайту рестрикції в ампліконі відбувається за рахунок використання особливого праймеру, який у своєму складі містить «помилковий» (mismatch) нуклеотид.…”