Allelic variants in alpha- and beta-globin genes of the river buffalo Bubalus bubalis L. as markers of useful genotype differentiation

The complex hemoglobin polymorphism of river buffalo (Bubalus bubalis L.) was addressed. Four - amplicons extending 1311 bp were obtained from the genome of river buffalo using primers designed on the basis of -globin gene sequences from goat; in the same way, five  amplicons extending from 1645 to 1803 bp were obtained using primers from -globin gene sequences from sheep. All amplicons were sequenced and the multiple alignments of the -globin genes pointed to a high degree of homology, the greatest difference being in intron 2 of a  allele called HBBA, containing a 119 bp insertion. Indeed, near to this region, all five -genes contained sequences identified as transposable elements, belonging to the family of short interspersed nuclear elements. Among the five genes, two were easily identified as alleles of adult - genes and called HBBA and HBBT, whereas the remaining genes were identified as embryonic (HBE), fetal (HBG) and pseudo gene (HB). Concerning the four -globin genes, the differences emerging allowed the characterization of the two haplotypes A and B even in river buffalo. In particular, haplotype A consists of two genes, called HBA1A and HBA2A, differing at codons 129 and 131, whereas haplotype B, with HBA1B and HBA2B, differs at codons 7, 10 and 11. Moreover, both genes of haplotype A differ from those of haplotype B at codon 64, where the triplet GCC for Ala changed to ACC for Asn. A multiple alignment definitively characterized their primary structures, thus contributing to highlight the different rate of gene homogenization. Finally, based on the alignments of the -globin genes among some ruminant species, a phylogenetic tree was obtained through the Neighbor-Joining method, in order to analyse the evolutionary events occurring in the species.