Complete CSN1S2 Characterization, Novel Allele Identification and Association With Milk Fatty Acid Composition in River Buffalo

Abstract

The αs2-casein is one of the phosphoproteins secreted in all ruminants' milk, and it is the most hydrophilic of all caseins. However, this important gene (CSN1S2) has not been characterized in detail in buffaloes with only two alleles detected (reported as alleles A and B), and no association studies with milk traits have been carried out unlike what has been achieved for other species of ruminants. In this study, we sequenced the whole gene of two Mediterranean river buffalo homozygotes for the presence/absence of the nucleotide C (g.7539G>C) realized at the donor splice site of exon 7 and, therefore, responsible for the skipping of the same exon at mRNA level (allele B). A high genetic variability was found all over the two sequenced CSN1S2 alleles. In particular, 74 polymorphic sites were found in introns, six in the promoter, and three SNPs in the coding region (g.11072C>T, g.12803A>T, and g.14067A>G) with two of them responsible for amino acid replacements. Considering this genetic diversity, those found in the database and the SNP at the donor splice site of exon 7, it is possible to deduce at least eight different alleles (CSN1S2 A, B, B1, B2, C, D, E, and F) responsible for seven different possible translations of the buffalo αs2-casein. Haplotype data analysis suggests an evolutionary pathway of buffalo CSN1S2 gene consistent with our proposal that the published allele CSN1S2 A is the ancestral αs2-CN form, and the B2 probably arises from interallelic recombination (single crossing) between the alleles D and B (or B1). The allele CSN1S2 C is of new identification, while CSN1S2 B, B1, and B2 are deleted alleles because all are characterized by the mutation g.7539G>C. Two SNPs (g.7539G>C and g.14067A>G) were genotyped in 747 Italian buffaloes, and major alleles had a relative frequency of 0.83 and 0.51, respectively. An association study between these SNPs and milk traits including fatty acid composition was carried out. The SNP g.14067A>G showed a significant association (P < 0.05) on the content of palmitic acid in buffalo milk, thus suggesting its use in marker-assisted selection programs aiming for the improvement of buffalo milk fatty acid composition.

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

“…These are classified into three main subclasses that include Termi-nal Inverted Repeat (TIR) DNA transposons, such as hATs and mariners; rolling-circle transposons, such as Helitrons; and self-synthesizing DNA transposons, such as Mavericks [49]. INEs have been extensively investigated in ruminants [13,52,53], but studies are also available for camelids CSN2 [19], CSN3 [23], and the entire casein cluster [3].…”

“…The sequenced regions included 752 bp at the 5 flanking region and 115/114 bp at the 3 flanking region and are identified in GenBank as ID numbers OQ730238 (C. bactrianus) and OQ730239 (C. dromedarius). The entire gene is approximately 15,000 bp long, with 14,735 bp in dromedary and 14,695 bp in Bactrian, consisting of 982 bp in exonic regions and about 14,000 bp in intronic regions (13,753 bp in dromedary and 13,713 bp in Bactrian). Exon 1 (48 bp) and the first 12 bp of exon 2 are noncoding.…”

“…In small domestic ruminants (goats and sheep), researchers have identified at least eight CSN1S2 alleles associated with three different αs2-CN content levels [7][8][9][10][11]. Among larger ruminants, cattle contain four variants (A, B, C, and D) [12], and the buffalo has eight alleles (A, B, B1, B2, C, D, E, and F) [13].…”

Novel Insight Into the Role of ACSL1 Gene in Milk Production Traits in Buffalo

“…Caseins are an important group of proteins in milk that accounted for approximately 80% of milk proteins and are secreted by the mammary epithelial cells ( Cavaletto et al, 2008 ). There are four types of casein: α s1 -casein, α s2 -casein, β-casein, and κ-casein, all of which possess different structures and functionality, and both α s1 -casein and β-casein are major caseins ( Cosenza et al, 2021 ). Wang et al (2014) found that Pten downregulates dairy cow mammary epithelial cell secretion of β-casein.…”

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

“…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.).…”