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Not AvailablePomegranate (Punica granatum L.) is an important fruit-bearing deciduous shrub which has been under cultivation since ancient times. Genomic resources like microsatellite markers are limited in pomegranate; therefore, this study was undertaken with the objective to develop and characterize microsatellite markers using Roche 454 GS FLX Titanium pyrosequencing technology. The total length of non-redundant sequences obtained was 19.7 Mbp, consisting of 59 603 reads assembled into 7361 contigs, of which, 567 (7.70%) contained microsatellite repeats. The average G+C content was 41.3%. Functional annotation to Arabidopsis thaliana sequences yielded 734 unigenes from 7361 contigs which were classified based on GO terms. Primer pairs were designed for total of 171 loci, out of which 167 produced reproducible polymorphic bands in 12 genotypes of pomegranate. A total of 951 alleles were identified, ranging from 1 to 14 per locus (average of 5.56). The polymorphism information content (PIC) value ranged between 0.00 and 0.910 (mean of 0.5427). The markers developed here will be useful for genetic diversity studies, gene mapping and genotyping in pomegranate.Not Availabl