Mitochondrial phylogenetic and diversity analysis in Azi-Kheli buffalo

Novelty statement The present study was conducted for the first time in Pakistan to investigate Cytochrome C Oxidase Subunit 1 (CO1) gene and full-length Displacement Loop (D-loop) region of mitochondrial DNA in Azi-Kheli buffalo breed native to northern hilly areas of Khyber Pakhtunkhwa Province of Pakistan. The present study was designed to investigate phylogeny and diversity in Azi-Kheli buffalo, through two mitochondrial DNA regions, i.e., Cytochrome C Oxidase Subunit-I (CO1) and Displacement Loop (D-loop) region. Thirty (30) blood samples were taken from Azi-Kheli pure breed animals from original breeding tract, i.e., Khwazakhela, Swat. Polymerase chain reactions using gene-specific primers were carried out for amplifying 709-bp region of CO1 gene and 1159-bp region of D-Loop for identification, phylogeny, and diversity in Azi-Kheli buffalo, respectively. The sequences of CO1 gene revealed four (04) haplotypes, whereas D-loop sequences revealed five (05) haplotypes. Mean interspecific diversity with related species was 2.56%, and mean intraspecific diversity within Azi-Kheli buffalo was 0.25%, estimated via Kimura-2 parameter. Phylogenetic tree (maximum likelihood) revealed clustering of Azi-Kheli haplotypes with river buffalo and is distinct from swamp buffalo and other related species of genus Bubalus. Mean haplotype and nucleotide diversity of D-loop were Hd = 0.9601 ± SD = 0.096 and π = 0.01208 ± SD = 0.00182, respectively. Phylogenetic tree (neighbor-joining) revealed two main clades, i.e., river buffalo and swamp buffalo clade. The haplotypes of Azi-Kheli clustered with haplotypes of different river buffalo breeds at different positions. The current study suggests that Azi-Kheli has common origin with other river buffalo breeds; hence, it is river buffalo which harbors high genetic diversity.