Development of polymorphic microsatellite loci for Iranian river buffalo (Bubalus bubalis )

Microsatellite loci were developed using PCR-based isolation of microsatellite arrays (PIMA) for Iranian river buffalo. Blood samples of eighty unrelated individuals from four buffalo populations (Khuzestan, Mazandaran, Guilan and Azarbayejan) were taken and following DNA extraction, isolation of microsatellite loci initiated using enrichment with random amplified polymorphic DNA (RAPD) primers. RAPD-PCR fragments were ligated into PTZ57R TA cloning vector and transformed into DH5
competent cells. Obtained colonies were screened for presence of repetitive elements by repeatspecific and M13 forward and reverse primers. After designing primer pairs for repeat containing fragments, they were tested in all buffalo populations. Two microsatellite loci (RBBSI and RBBSII) were informative and polymorphic. Number of alleles for RBBSI and RBBSII in 80 individuals was 5 and 6, respectively. Expected heterozygosity ranged from 0.65 to 0.81. Significant deviation from Hardy-Weinberg equilibrium expectation occurred for both loci in all populations, but 37.5% of locus/population combination showed the deviation. We postulate that the two newly isolated microsatellite loci during this study could be useful for population genetic studies in Bubalus bubalis.