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The Hakka, who have undergone a series of great migrations, are usually identified with people who speak the Hakka language or share at least same Hakka ancestry. As the largest Hakka dialect island in West China, the Dongshan region was closely linked with the great migration wave of Hakka. However, the paternal genetic profiles of Dongshan Hakka have never been revealed. In the present study, 41 Y-chromosomal short tandem repeat (Y-STR) loci included in the SureID PathFinder Plus Kit were analyzed in 353 unrelated male individuals (171 Hakka and 182 Han) of Sichuan Province, China. By analyzing 166 different haplotypes among Dongshan Hakka and 176 different haplotypes among Sichuan Han males, haplotype diversity was calculated as 0.9997, with a discrimination capacity of 0.9708, for the Hakka population, and 0.9996 and 0.9670 for the Sichuan Han population, respectively. Most of the Y-STR loci were highly informative in both populations except DYS645. The genetic relationships were evaluated by comparing the Hakka population with 11 other groups that are relevant to Hakka migration routes. The results of the multidimensional scaling plot and phylogenetic tree indicate that the Dongshan Hakka population was closely related to Han nationalities from Anhui, Jiangxi, and Fujian Provinces.