Molecular Characterization of the Bovine CSN3 and LGB Milk Protein Genes Using Sequencing and PCR-RFLP Markers in Ethiopian Indigenous Cattle Populations

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Addis Ababa University


In the last few decades, genetic polymorphism of bovine milk protein genes such as caseins and whey proteins has achieved considerable research interest mainly due to their significant associations with fat and protein contents as well as the manufacturing properties of milk. In this study, genetic polymorphisms of two bovine milk protein genes namely CSN3 and LGB were analyzed in five indigenous and one crossbred cattle breeds using PCR-RFLP markers and sequencing. Moreover, the magnitude of genetic variability within and among these cattle populations at these two loci was estimated hence their population structure and phylogenetic relationships characterized. For this purpose, genomic DNA was extracted from 83 animals belonging to Abigar, Boran, Guraghe, Horro, Sheko and Holestin-Barka crossbred cattle populations. Both of the markers used to analyze polymorphisms in the CSN3 and LGB genes were informative and enabled us to detect the widely reported CSN3-A and B and LGB-A and B variants. Furthermore, sequencing revealed additional genotypes among which were the zebuspecific CSN3-AI and H haplotypes observed in 50% of the sequences from the indigenous cattle and CSN3-E in one sequence derived from a Holstein-Barka crossbred cow. These CSN3 haplotypes were deduced analyzing a total of 7 point mutations found in the 633bp region sequenced of which 5 were base transitions and 2 transversions. Two of these sites were silent substitutions while the rest were non-synonymous. At LGB locus, 12 SNPs were detected in the amplified 529 bp region where only 3 resided in the coding region corresponding to 2 silent and one non-synonymous substitution. Analysis of these mutations yielded two new haplotypes (LGBB 1 and B*) that where results of silent mutations. The PCR-RFLP assay revealed that CSN3-A and LGB-B were more prevalent than CSN3-B and LGB-A variants in the indigenous cattle population investigated. The frequency of allele CSN3-A (0.687) was more than twice that of B (0.313) where as LGB-B had a very high frequency of 0.861 compared to LGB-A (0.139). All of the breeds met HWE at both loci with the exception of Sheko that deviated significantly (P<0.05) at the LGB locus. The total population revealed a rather moderate level of genetic variability (0.34). The highest variability was observed in Sheko (0.414) while the lowest was in Guraghe (0.273). When the two loci were considered separately, CSN3 locus presented higher (43.04%) level of gene diversity compared with LGB locus (24.42%) which was rather more homogenous. A higher level of inbreeding was detected at LGB locus (FIS=0.173) compared with CSN3 which instead showed excess heterozygosity with FIS of -0.0282. When considering both loci, the mean FIS and FIT estimates were 0.044 and 0.07 respectively. The overall FST value (0.028) revealed little and insignificant (P>0.05) genetic differentiation among the populations studied. Most of the total genetic variation was attributed to the within population (97.23%) whereas little and insignificant level of variation was witnessed among the different cattle breeds (2.77%). Pairwise genetic distances revealed very small distances among the breeds studied. However, Sheko had larger distances when paired with each breed where the maximum of which was with Abigar and the minimum that involving Guraghe indicating its relative departure from the rest. Similarly, the dendrogram generated clearly depicted Sheko forming a separate branch where the rest shared a second main branch. Key words: PCR-RFLP, Sequencing, diversity, Ethiopian cattle, SNPs, CSN3, LGB, HWE



PCR-RFLP, Sequencing, diversity, Ethiopian cattle, SNPs, CSN3, LGB, HWE