Y-chromosomal microsatellites (Y-STRs) are widely used to infer population histories, discover genealogical relationships, and identify males for criminal justice purposes. Buccal swabs samples were collected from unrelated Yousafzai donors living in and around Swat district of Khyber Pakhtoonkhwa, Pakistan. DNA was extracted, quantified by the Nanodrop-1000, and genotyped using the AmpFlSTR® Yfiler™ kit (Applied Biosystems). Allele frequencies were calculated using the gene count method. Statistical analysis was performed and as many as 35 haplotypes were identified and 30 of them were observed only once, accounting for 90.47% unique haplotypes. The most common haplotypes were observed sixteen times (26.2%). Y chromosomal haplotypes diversity for 17 Y STRs locus set ranged from 0.155 to 0.591 in Yousufzai population indicating haplotypes among unrelated males. The highest locus diversity value was 0.591 at locus DYS391 while the lowest locus diversity value was 0.155 for DYS456 locus. Multidimensional scaling analysis (MDS) plot was drawn based on the Fst value. All populations showed a high level of haplotype diversity, with low inter-population variance as measured by an analysis of molecular variance. The Yousafzai allele frequency database was then compared with other populations of the world.
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