Detection of A2A2 genotype of beta casein protein (CSN2) gene in local, exotic and cross bred cattle in Pakistan.

Genetic variants of bovine Beta-casein protein (CSN2) gene especially A1 and A2 are the most important variants in dairy cattle. A1 milk protein is considered as risk factor for different disease and milk intolerance which release Beta-Casomorphin-7 during digestion which is a bioactive opioid but not released from A2 milk protein. This opioid is responsible for several human health problems like Coronary Heart disease, type 1 diabetics, milk intolerance and other neurological disorders. In present study, 360 blood sample were collected from Lohani, Achai, jersey, Holstein Friesian, Achai x jersey, Friesian x Sahiwal and Sahiwal x Friesian from different region of Khyber Pakhtunkhwa (KP) province. The polymerase chain reaction (PCR) amplicons were sequenced for the identification of polymorphism in exon 7 of Beta-casein protein (CSN2) gene. Sequencing analysis explored CSN2 genotype in exon 7 using the Genomic sequence from GenBank (X.71104) g.8101 C > A at codon 67. The allelic and genotypic frequencies of CSN2 gene were analyzed and observed that Holstein Friesian cattle exhibited A1A2 33%, A1A1 50% and A2A2 17%, Jersey cattle show 68% A1A1, 18% A1A2 and 14% A2A2, Sahiwal x Friesian 56% A1A1, 26% A1A2 and 18% A2A2, Jersey × Achai 78% A2A2, 15% A1A2 and 7% A1A1, Achai 100% A2A2 Lohani 100% A2A2. This is a preliminary study, conducted with meager resources, therefore, it is very difficult to make conclusion that which particular breed possess harmful alleles and which breed possess useful alleles of beta-casein gene. Therefore, a comprehensive molecular work is needed to be performed with greater number of samples sequencing.

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.