The CSN1S2 gene encodes αs2-casein, the third most abundant protein in camel milk. Despite its importance in foals, human nutrition, and dairy processing, the CSN1S2 gene in camels has received little attention. This study presents the first complete characterization of the CSN1S2 gene sequence in Old-World camels (Camelus bactrianus and Camelus dromedarius). Additionally, the gene promoter, consisting of 752 bp upstream of exon 1, was analyzed. The entire gene comprises 17 exons, ranging in length from 24 bp (exons 4, 8, 11, and 13) to 280 bp (exon 17). Interesting was the identification of the exon 12 in both species. The promoter analysis revealed 24 putative binding sites in the Bactrian camel and 22 in dromedary camel. Most of these sites were typical elements associated with milk protein, such as C/EBP-α, C/EBP-β, Oct-1, and AP1. The SNP discovery showed relatively high genetic diversity compared to other camel casein genes (CSN1S1, CSN2, and CSN3), with a total of 34 polymorphic sites across the two species. Particularly noteworthy is the transition g.311G>A in the CSN1S2 promoter, creating a new putative consensus binding site for a C/EBP-β in the Bactrian camel. At the exon level, two novel variants were found. One was detected in exon 6 of the Bactrian camel (g.3639C>G), resulting in an amino acid replacement, p.36Ile>Met. The second variant was found in noncoding exon 17 of dromedary CSN1S2 (g.1511G>T). Although this mutation occurs in the 3′-UnTranslated Region, it represents the first example of exonic polymorphism in the CSN1S2 for this species. This SNP also affects the binding sites of different microRNAs, including the seed sequence of the miRNA 4662a-3p, highlighting its role as a regulatory factor for CSN1S2 gene. A PCR-RFLP was set up for genotyping a dromedary Tunisian population (n = 157), and the minor allele frequency was found to be 0.27 for the G allele, indicating a potential yield improvement margin. The interspersed elements (INEs) analysis revealed 10 INEs covering 7.34% and 8.14% of the CSN1S2 sequence in the Bactrian and dromedary camels, respectively. Furthermore, six elements (A, B, F, H, I, and L) are shared among cattle and camels and are partially found in other ruminants, suggesting a common ancestral origin of these retrotransposons. Conversely, elements C, D, E, and G are specific to camels.

Background/Objectives: Buffalo populations exhibit distinct genetic variations influenced by domestication history, geographic distribution, and selection pressures. This study investigates the genetic structure and differentiation of 11 buffalo populations, focusing on five loci related to milk protein (CSN1S1 and CSN3) and fat metabolism (LPL, DGAT1 and SCD). The aim is to assess genetic variation between river, swamp, and wild-type buffaloes and identify key loci contributing to population differentiation. Methods: Genetic diversity was analyzed through allele frequency distribution, the Hardy−Weinberg equilibrium testing, and observed (Ho) and expected heterozygosity (He) calculations. Population structure was assessed using principal component analysis (PCA), F_ST statistics, and phylogenetic clustering (k-means and UPGMA tree). The silhouette score (SS) and the Davies−Bouldin index (DBI) were applied to determine optimal population clustering. Results: Significant genetic differentiation was observed between river and swamp buffaloes (p < 0.001). DGAT1 and CSN3 emerged as key markers distinguishing buffalo types. The Italian Mediterranean buffalo exhibited the highest genetic diversity (Ho = 0.464; He = 0.454), while the Indonesian, Chinese, and Vietnamese populations showed low heterozygosity, likely due to selection pressures and geographic isolation. The global F_ST (0.2143; p = 0.001) confirmed moderate differentiation, with closely related populations (e.g., Nepal and Pakistan) exhibiting minimal genetic divergence, while distant populations (e.g., Egypt and Indonesia) showed marked differences, and the Romanian population showed a unique genetic position. Conclusions: These findings contribute to a deeper understanding of buffalo genetic diversity and provide a valuable basis for exploiting the potential of this species in the light of future breeding and conservation strategies specific for each buffalo type.

Alpha-Lactalbumin (alpha-LA), which is encoded by the LALBA gene, is a major whey protein that binds to Ca2+ and facilitates lactose synthesis as a regulatory subunit of the synthase enzyme complex. In addition, it has been shown to play central roles in immune modulation, cell-growth regulation, and antimicrobial activity. In this study, a multitechnical approach was used to fully characterize the LALBA gene and its variants in both coding and regulatory regions for domestic camelids (dromedary, Bactrian camel, alpaca, and llama). The gene analysis revealed a conserved structure among the camelids, but a slight difference in size (2,012 bp on average) due to intronic variations. Promoters were characterized for the transcription factor binding sites (11 found in total). Intraspecies sequence comparison showed 36 SNPs in total (2 in the dromedary, none in the Bactrian camel, 22 in the alpaca, and 12 in the llama), whereas interspecies comparison showed 86 additional polymorphic sites. Eight SNPs were identified as trans-specific polymorphisms, and 2 of them (g.112A>G and g.1229A>G) were particularly interesting in the New World camels. The first creates a new binding site for transcription factor SP1. An enhancing effect of the g.112G variant on the expression was demonstrated by 3 independent pGL3 gene reporter assays. The latter is responsible for the p.78Ile>Val AA replacement and represents novel allelic variants (named LALBA A and B). A link to protein variants has been established by isoelectric focusing (IEF), and bioinformatics analysis revealed that carriers of valine (g.1229G) have a higher glycosylation rate. Genotyping methods based on restriction fragment length polymorphism (PCR-RFLP) were set up for both SNPs. Overall, adenine was more frequent (0.54 and 0.76) at both loci. Four haplotypes were found, and the AA and GA were the most common with a frequency of 0.403 and 0.365, respectively. Conversely, a putative biological gain characterizes the haplotype GG. Therefore, opportunities for rapid directional selection can be realized if this haplotype is associated with favorable milk protein properties. This study adds knowledge at the gene and protein level for alpha-LA (LALBA) in camelids and importantly contributes to a relatively unexplored research area in these species.