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In this study, 30 microsatellite markers recommended by the International Society for Animal Genetics and the Food and Agriculture Organization were used to determine the extent of genetic differentiation and phylogenetic relationships among indigenous chicken populations sampled in Kenya, Uganda, Ethiopia and Sudan. Genetic differentiation (FST) and chord genetic distances (DC) indicated that the indigenous chickens were genetically related but distinct from commercial broiler and egg layer lines. Genetic divergence among the indigenous chickens determined using the Mantel test was significantly influenced (P < 0.001) by geographic (reproductive) isolation. Genetic subdivisions were found between the Kenyan/Ugandan chicken populations and Ethiopian/Sudanese chicken populations. The Marsabit chicken population from northern Kenya was the most genetically distinct population within the Kenyan and Ugandan chicken cluster, thus warranting further investigation.