New MTCYB haplotypes in Romanian buffalo.

The Romanian Buffalo Breed (RBB), established in 1987, originated from local river buffalo populations highly adapted to the low-input breeding condition. Cross-breeding with Asian breeds was recently used for genetic improvement in milk production. In the last 25 years the species has dramatically decreased. Over 90% of livestock has been sold and never replaced. The population is now mainly maintained in subsistence farms. This study aims to assess the genetic diversity within the remaining population and to verify links with improved breeds supposedly introgressed in RBB. To assess the genetic diversity of RBB and ascertain possible phylogenetic relations with other buffalo breeds, we sequenced the entire cytochrome B gene (MTCYB) in a sample population. Blood samples were collected from randomly selected 52 unrelated individuals from various locations in Transylvania. A DNA fragment containing the entire MTCYB gene (1140bp) was amplified by PCR directly from whole blood (1μl). The amplicons were sequenced using two pairs of primers. The sequences were subsequently used for genetic diversity assessments. Analysis of the sequences led to the identification of five novel MTCYB haplotypes, uploaded in GenBank with the following accession numbers: JQ241279; JQ241280; JQ241281; JQ241282; JQ241283. In the sample population, the most frequently identified haplotypes were haplotype 2 (EF409940.1) and haplotype 3 (EF409941.1), previously reported in other buffalo populations, mainly from Asia, confirming the Asiatic origins of RBB. Sequence comparisons have revealed that RBB is mainly related with Indian breeds. Despite the numerical decline of RBB, through correct breeding schemes the breed maintained a good level of genetic diversity. Our analysis confirms RBB’s Asiatic origins. Presence of new haplotypes may also reflect that this population is isolated from a geographical and reproductive point of view.