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

Gianfranco Cosenza,Daniela Gallo,Giustino Gaspa,Barbara Auzino,Alfredo Pauciullo

doi:10.3389/fgene.2020.622494

Gianfranco Cosenza, Daniela Gallo + Show 3 more

Open Access

https://doi.org/10.3389/fgene.2020.622494

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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.

Highlights

The αs2-casein (207 aa) is one of the phosphoproteins secreted in ruminants’ milk in the form of stable calcium–phosphate micelles, and it is the most hydrophilic of all caseins
Goat and sheep showed a higher level of genetic diversity at CSN1S2, and nowadays, at least seven alleles associated with three different αs2-CN levels have been characterized in both species (Boisnard et al, 1991; Ramunno et al, 2001a,b; Giambra and Erhardt, 2011)
By using genomic DNA as template, we sequenced the whole gene encoding the αs2-casein (CSN1S2) of two Mediterranean river buffalo homozygotes for the presence/absence of the nucleotide C (FM865620:g.773G>C) that caused inactivation of the intron 7 splice donor site and, the allele-specific exon skipping characteristic of the CSN1S2 B allele

Summary

Introduction

The αs2-casein (207 aa) is one of the phosphoproteins (αs, β, αs, and k) secreted in ruminants’ milk in the form of stable calcium–phosphate micelles, and it is the most hydrophilic of all caseins. The molecular weight of this protein was assessed to be 22,741 Da in buffalo vs 25,226 in cattle (Feligini et al, 2009). The αs2CN is the third most abundant casein fraction (4.99 g/L), and the corresponding coding gene (CSN1S2) showed a lower translation efficiency (0.25) compared to the other casein genes as CSN3 (k-CN, 2.69), CSN2 (β-CN, 2.39), and CSN1S1 (αs1-CN, 1.31) (Cosenza et al, 2011). Goat and sheep showed a higher level of genetic diversity at CSN1S2, and nowadays, at least seven alleles associated with three different αs2-CN levels have been characterized in both species (Boisnard et al, 1991; Ramunno et al, 2001a,b; Giambra and Erhardt, 2011). The alleles B and C are specific for the zebu and yak cattle, respectively (Ibeagha-Awemu et al, 2007)

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