Genetic characterization of male lineages of Pathans from Pakistan

Hwan Young Lee1 · Na Young Kim1 ·JeongEunSim1 · Ajin Choi1 · Allah Rakha2 · Myung Jin Park1 · Ja Hyun An1 ·Woo IckYang1 · Kyoung-Jin Shin1

1Department of Forensic Medicine, Yonsei University College of Medicine, Seoul, Korea 2National Forensic Science Agency, National Police Bureau, Islamabad, Pakistan

Introduction Cultural and linguistic affiliations divide the people of Pakistan into 16 ethnic groups with diverse origins. The evolutionary antiquity and endogamy of Pakistani populations generate a high degree of genetic differentiation and structuring. Hence to obtain the most reliable and conservative frequency estimates for forensic purposes and to estimate population history require that regional or ethnic databases be established. Major ethnic groups of Pakistan include the Punjabis, Pathans, Sindhis, Seraikis, Muhajirs, Balochis, Hindkowans, and Chitralis. The Pathans represent the tribes who speak Pashto (Eastern Iranian branch of the Indo‐Iranian language family), inhabit mainly the North West Frontier Province (N.W.F.P.), adjoining tribal areas of Pakistan, and southern and eastern parts of Afghanistan, and are the second‐largest ethnic group in Pakistan. In the present study, to better characterize and understand the male lineages of Pakistani populations, 22 Y‐STRs and 18 Y‐SNPs were analyzed in 270 unrelated Pathans from the N.W.F.P. and Federally Administered Tribal Areas (FATA) of Pakistan. Materials and Methods Samples Y chromosomal haplogroup determination using multiplex SBE Blood samples were collected from 270 unrelated male Pathan volunteers in Y chromosomal SNP selection and primer design for PCR and SBE the N.W.F.P and FATA of Pakistan. All participants gave their informed consent Fifteen (M40, M89, M201, M69, M304, M9, M20, M45, M242, M207, P231, orally or in writing after we explained the aims and procedures of the study. M17, M479, M124 and M184) and 3 Y‐SNPs (RPS4Y711, JST002611 and M117) The Institutional Review Board of Severance Hospital, Yonsei University in were selected to determine Y‐chromosomal haplogroups E, F, G, H, J, K, L, P, Seoul, Korea approved this study. DNA was isolated from blood using QIAamp Q, R, R1, R1a1a, R2, R2a and T, and C, O3a1c and O3a2c1a, that are present in DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s South and East Asian populations, respectively. Primers for PCR amplification instructions. and subsequent SBE were designed using programs Primer 3 Schematic of 3 in‐house multiplex PCRs for analysis of 22 Y‐STRs (http://frodo.wi.mit.edu/primer3/) and Batchprimer 3 (http://batchprimer3. bioinformatics.ucdavis.edu/index.html). 100 bp 150 bp 200 bp 250 bp 300 bp Multiplex PCR and multiplex SBE 392 389I 19 389II Multiplex PCR: Several multiplex and monoplex PCRs were performed in a I 93-129 142-174 175-211 254-298 393 391 390 385 final volume of 25 lthat contained 1 ng of template DNA, 2.5 lof Gold ST*R 100-140 144-180 188-236 241-305 10× buffer (Promega, Madison, WI, USA), 2.0 U (1.0 U for monoplex) of

H4 439 635 448 AmpliTaq Gold® DNA polymerase (Applied Biosystems) and primer mix. II 122-146 202-234 247-279 299-341 Thermal cycling was performed under the following conditions: 95°C for 11 456 458 437 385 438 ° ° ° 91-115 133-165 182-198 241-305 311-346 min; 33 cycles of 94 C for 20 sec, 60 C for 1 min, 72 C for 30 sec; and a final extension of 72°C for 7 min. For the following SBE, 5.0 lof PCR products was 385 464 168-232 248-288 purified with ExoSAP‐IT (USB, Cleveland, OH, USA). III 388 447 Multiplex SBE: Several multiplex and monoplex SBE reactions were carried out 123-138 187-242 TM 446 449 with the SNaPshot Multiplex kit (Applied Biosystems) according to the 137-177 222-258 manufacturer’s instruction. After the SBE reactions, 1 U SAP (USB) was added The 22 Y‐STRs were amplified using AmpFlSTR® YfilerTM PCR amplification kit to the extension products, and the mix was incubated at 37°C for 45 min (Applied Biosystems, Foster City, CA, USA) and an in‐house multiplex PCR followed at 80°C for 15 min to remove the unincorporated ddNTPs. system (Please refer to http://forensic.yonsei.ac.kr/protocols.html). Detection system: ABI PRISM 310 Genetic Analyzer and GeneMapper ID 3.2 Haplotype diversity was calculated with the software package Arlequin 3.5.1.2 software (Applied Biosystems)

Results Fig. 1. Phylogenetic tree of the 18 Y‐ Fig. 2. Representative electropherograms of the Table 1. Y‐STR haplotype diversities and chromosomal binary polymorphisms and with multiplex SBE reactions for Y‐haplogrouping of discrimination capacities in each haplogroup their corresponding haplogroup frequencies Pathans from Pakistan observed in Pathans from Pakistan observed in Pathans from Pakistan Haplogroup G Haplogroup No. Haplotype Discrimination Haplogroup N Frequency M40 M89 M201 M69 M304 M9 diversity capacity* A,B A,B DE (xE) C 2 1.0000 1.0000 DE M40 E E RPS4Y C C 2 0.0074 G 38 0.9829 0.8421 F (xG, H, K) F M89 M201 G G 38 0.1407 H 14 1.0000 1.0000 M69 Haplogroup J H H 14 0.0519 M40 M89 M201 M69 M304 M9 J 26 0.9815 0.8077 IJ (xI, J) I I M304 K 1 ‐ ‐ J J 26 0.0963 K* 1 0.0037 K M9 L M20 L 16 0.0593 L 16 0.9917 0.9375 M M NO* Haplogroup R1a1a O3a1c 1 ‐ ‐ N N O O JST002611, M133 M20 M45 M242 M207 P231 M17 O3a‐sub 2 0.0074 O3a2c1a 1 ‐ ‐ P M14 Q M242 Q 14 0.0519 Q 14 0.9890 0.9286 R* R1* R1a1a 133 0.9892 0.7970 a R1a* P231 R M207 1 R1a1* a M17 Haplogroup R2a R2 1 ‐ ‐ 1 R1a1a 133 0.4926 b R1a1b c R1a1c M20 M45 M242 M207 P231 M17 R2a 19 1.0000 1.0000 d R1a1d e R1a1e T 4 1.0000 1.0000 b R1b

2 M479 R2 (xR2a) 1 0.0037 Total 270 0.9968 0.8519 a M124 R2a 19 0.0704 S S M479 M124 T M184 T 4 0.0148 * Discrimination capacity was determined by dividing the number of observed haplotypes by the number of total investigated individuals. Discussion . In haplotype analysis for the 22 Y‐STRs in 270 Pathan males, 230 different haplotypes were observed with overall haplotype diversity of 0.9968. Among them, 211 (78.1%) were observed once; 13 (9.6%), twice; 2 (2.2%), three times; 3 (5.6%), five times; and one (4.4%) twelve times. The relatively low values for haplotype diversity and discrimination capacity in Pathans should be considered in the forensic interpretation of Y‐STR data. . With the developed multiplex SBE reactions and additional SNP analysis, 13 different Y‐chromosomal haplogroups were defined within our samples, and the R1a1a haplogroup of South Asian origin was most frequently observed. . Haplogroup distribution in our samples is mainly consistent with the previous study by Firasat et al. (EJHG 15:121), but to better characterize and compare the male lineages of Pakistani populations, further investigation of the other ethnic groups will be needed.