Investigation on genetic structure of the Iranian buffalo by microsatellite markers.

In order to study phylogenetic relationship of the Iranian buffalo population, 360 blood samples were collected from North (Mazandaran & Guilan), Khuzestan and Azerbaijan and DNA was extracted using salting out and Kit methods. Fifteen microsatellite markers, CSSM019, CSSM029, CSSM033, CSSM038, CSSM041, CSSM043, CSSM047, CSSM057, CSSM060, CSRM061, CSSM062, CSSM070, BRN, BMC1013 and ETH003 were selected according to their polymorphism and they were amplified after PCR optimization. PCR products were run on denaturized Acryl Amid gels and genotype of the animals, was detected after silver staining method. All markers were significantly deviated from Hardy-Weinberg equilibrium (P0.001). CSSM057 was not amplified. CSSM033, CSSM047, CSSM062 and CSSM070 were the best and CSSM029 was the worst in all populations according to their polymorphism information content. The mean of observed and effective number of alleles has been higher in the Azerbaijan buffalo population (4.36 and 3.52) than Khuzestan (4.28 and 3.32) and North Iranian buffalo (4.21 and 3.31) populations. The mean of observed and effective number of alleles were equal to 4.36 and 3.74 respectively in all Iranian buffalo population. The mean of expected heterozygosity in North, Azerbaijan, Khuzestan and all Iranian buffalo population were equal to 0.68, 0.69, 0.68 and 0.71 respectively. It shows the high level of polymorphism in the Iranian buffalo population. The Shannon's information index of the Iranian buffalo population was equal to 1.34 ± 0.31 and it has been higher in Azerbaijan (1.31 ± 0.29) than North (1.25 ± 0.25) and Khuzestan (1.25 ± 0.28) buffalo populations. The mean of polymorphism information content of the Azerbaijan (0.64 ± 0.03) has also been higher than North (0.62 ± 0.03) and Khuzestan (0.62 ± 0.03) buffalo populations. The mean of Fis (related to inbreeding coefficient) was equal to -30.7 in Azerbaijan, Khuzestan and North subpopulation according to the observed and expected heterozygosity. It (Fit) was equal to -26.7 in all Iranian buffalo population and shows enough genetic variation in the above population. The value of Fst (0.034) showed a little difference between all subpopulation of the Iranian buffalo. The correlation of the genetic distance measurement methods (Nei, Cavalli-Sforza and Edwards and Rynold, Weir and Cockerham) that were used in this research was 98.41 to 99.95 and the lowest and highest genetic distance was observed between North-Azerbaijan and North-Khuzestan respectively. The results of the genetic similarity, estimated by total allelic relationship method (TAR), were showed the highest genetic similarity between North and Azerbaijan (58.5) and the lowest between North and Khuzestan (53.8) buffalo population respectively. The most and the least genotypic similarity, were also observed between animals of the Khuzestan (64.4) and the Azerbaijan (60.8) buffalo population according to the results of the TAR method. The amount of genetic variation within population (0.88 to 0.90) was significantly greater than genetic variation between populations (0.10 to 0.12) according to the analysis of the molecular variance. The genetic variation between geographical regions (North and western south), between population within regions (North and Azerbaijan buffalo population in Northern region and Khuzestan buffalo population in western south region) and within population was equal to 0.11, 0.03 and 0.86 respectively according to the analysis of the molecular variance (P0.05). Phylogenetic tree based on Nei distances were drown using the neighbor-joining (NJ) and unweighted pair-group method using an arithmetic average (UPGMA). With both methods, the Northern and Azerbaijan buffalo population were located together at one cluster and Khuzestan buffalo population at the other cluster. Bayesian method (accuracy 85 percent) was the best between all likelihood and distance based methods in assignment test.