- Email: [email protected]
Population genetics of 17 Y-STR markers in Turkish Cypriots from Cyprus
Forensic Science International: Genetics 10 (2014) e1–e3
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Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Forensic Population Genetics - Short Communication
Population genetics of 17 Y-STR markers in Turkish Cypriots from Cyprus§ K. Teralı a, T. Zorlu b, O. Bulbul b, C. Gurkan a,* a b
Turkish Cypriot DNA Laboratory, Committee on Missing Persons in Cyprus Turkish Cypriot Member Office, Nicosia, North Cyprus Institute of Forensic Science, Istanbul University, Istanbul, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 October 2013 Received in revised form 7 January 2014 Accepted 8 January 2014
We analyzed seventeen Y-chromosomal short tandem repeats (STRs) [DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y-GATA-H4, DYS437, DYS438, and DYS448] in 253 unrelated, male individuals from the Turkish Cypriot population of the Eastern Mediterranean island of Cyprus. While 206 out of the 253 haplotypes present in the dataset were unique, there are also 22 haplotypes that were observed in two individuals each, and 1 haplotype that was observed in three individuals. While no locus duplications or null alleles were observed in our dataset, we have detected 43 allelic variants in total, the majority of which (25 out of 253 haplotypes or 9.88%) comprised of .2 intermediate variants at the DYS458 locus (alleles 16.2, 17.2, 18.2, 19.2, and 20.2). For the 229 different haplotypes observed in the Turkish Cypriot dataset, the calculated discrimination capacity (DC) was 0.9051 and the haplotype diversity (HD) was 0.9992. The calculated average gene diversity (GD) values ranged from 0.3828 to 0.9631 for the DYS392 and DYS385a/b loci, respectively. Pairwise genetic distance comparisons of the Turkish Cypriot Y-STR dataset with those from the neighbouring (Turkey, Greece, Israel/Palestinian Authority area, Egypt and Italy) and relatively distant (Lithuania, Taiwan and Australia) countries through the use of analysis of molecular variance (AMOVA) and multi-dimensional scaling (MDS) analyses confirmed that our data do not deviate significantly from the typical core haplotypes of the Eastern Mediterranean region. The Turkish Cypriot Y-STR haplotype dataset will find an immediate use in the Committee on Missing Persons in Cyprus Project on the ‘‘Exhumation, Identification and Return of Remains of Missing Persons’’ and it is also expected to contribute to the establishment of forensic genetic services in North Cyprus. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Y-filer Y-STR Short tandem repeats (STRs) Cyprus Turkish Cypriot
1. Population Cyprus is the third largest island in the Mediterranean Sea situated 75 km to the South of Turkey, 200 km to the North West of Israel/Palestinian Authority area, 380 km to the North of Egypt, and 800 km to the South East of the Greek mainland. The earliest confirmed site of human activity on the island dates back to around 12,000 years before present [1]. Strategically situated at the crossroads of major civilizations and historical population movements, Cyprus subsequently received waves of ancient Greek settlements and Phoenician colonizations, ruled by the Assyrian, Persian, Hellenistic, Roman and Byzantine Empires, the latter of which was also intermitted with Arab conquests and an ArabByzantine condominium, taken over by the Crusaders/Lusignans/
§ Access to data is available upon request from the corresponding author ([email protected]). * Corresponding author. Tel.: +90 542 8584783; fax: +90 392 2238856. E-mail address: [email protected] (C. Gurkan).
1872-4973/$ – see front matter ß 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigen.2014.01.003
Venetians, conquered by the Ottoman Empire and finally occupied by the British Empire prior to her independence in 1960. Today, there are two major ethnic groups on the island, namely the Turkish Cypriots who speak Turkish and are Muslims and the Greek Cypriots who speak Greek and are mostly Orthodox Christians. There are also other constitutionally recognized ethnic groups that include Maronites, Armenians, and Latins. The subject of this study, the Turkish Cypriot population, resided all over the island in villages or cities wherein the populace comprised of either mixed ethnic background or exclusively Turkish Cypriot up until 1963/1974, but has largely relocated/concentrated in the Turkish Cypriot administrated North Cyprus since 1974. 2. Sampling Blood or buccal swab samples were collected along with signed informed consents from 253 unrelated, male individuals from the Turkish Cypriot population currently residing in North Cyprus. The participants also filled out a brief survey on their ethnic origin,
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K. Teralı et al. / Forensic Science International: Genetics 10 (2014) e1–e3
which included questions on their birthplace and that of their parents, as well as any other additional information that they may have with respect to their paternal pedigree. For the purposes of this study, we aimed to select each participant among volunteers who were born in Cyprus and/or currently residing in North Cyprus, but also born to Turkish Cypriot parents, whereby for the large majority of cases both father and mother were born in Cyprus, and if not, at least the father was born in Cyprus. Considering the relatively recent mass population movements of the Turkish Cypriots across the island over the last fifty years or so as described above, the traditional paternal birthplace of the volunteers were assumed as the origin of each given Y-STR haplotype (Ht) observed. 3. DNA extraction Genomic DNA extraction was carried out using the PureLinkTM Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. 4. PCR amplification PCR for 17 Y-STR loci [DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y-GATA-H4, DYS437, DYS438, and DYS448] was performed in a multiplex reaction using the AmpFlSTR1 Y-filerTM PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA). The total amplification reaction volume was 25 ml, which contained 9.2 ml of reaction mix, 5.0 ml of primer set, 0.8 ml of AmpliTaq Gold DNA Polymerase and 10 ml of genomic DNA (approximately 0.5– 1 ng ml 1). Amplifications were performed as described in the instruction manual of the AmpFlSTR1 Y-filer TM PCR Amplification Kit and using the Applied Biosystems 2720 Thermal Cycler (Applied Biosystems) [2]. 5. Electrophoresis and typing Separation and detection of the 17 polymorphic Y-chromosome-specific PCR amplicons were performed using the ABI 3130 Genetic Analyzer (Applied Biosystems). Each sample was prepared by adding 1.5 ml of PCR product to a mixture of 8.4 ml of Hi-DiTM formamide and 0.6 ml of GeneScanTM-500 LIZTM Internal Size Standard (both Applied Biosystems). Samples were analyzed using GeneMapper v4.0 (Applied Biosystems). Alleles were assigned according to the current International Society of Forensic Genetics (ISFG) guidelines for forensic Y-STR analysis [3]. 6. Data analyses Among the 253 haplotypes observed in the Turkish Cypriot YSTR dataset, 22 haplotypes were each detected in two individuals and 1 haplotype was detected in three individuals (Table S1). Notably, 2 out of the 3 individuals who share the same haplotype that was observed in triplicate (Ht 035), as well as 7 out of the 22 pairs of individuals who share the same haplotype in each pair (Hts 066, 070, 079, 087, 142, 166, and 193), were also found to share the same paternal geographic origins (i.e., small, isolated villages) in each pair, albeit without any evidence toward close paternal relatedness. In other words, a total of 8 pairs of seemingly unrelated individuals were found to share not only the same haplotype in each pair, but also the same paternal geographic origin. These observations perhaps could be attributed to a past tendency toward patrilocal residence patterns in isolated subpopulations of Turkish Cypriots. The Turkish Cypriot Y-STR dataset has 206 unique haplotypes (UH) (81.42%; number of unique haplotypes observed/total number of samples analyzed 100) and
229 different haplotypes, thus yielding a very high discrimination capacity (DC) (0.9051, total number of different haplotypes observed/total number of samples analyzed). The most common haplotype observed in the dataset is found at a frequency of 1.19% (Ht 035, observed in triplicate), which has so far never been reported in the YHRD database (as of 22 August 2013) [4]. While we did not observe any locus duplications or null alleles in the 253 samples analyzed, based on the observed fragment sizes, we observed 43 allelic variants in total. 25 of the allelic variants (9.88% of all haplotypes) we observed were .2 intermediate alleles at the DYS458 locus, namely 16.2, 17.2, 18.2, 19.2 and 20.2. Presence of this relatively high percentage of DYS458*.2 variants (9.88%) in the Turkish Cypriot population compares well with those from geographically and/or historically related countries, such as Italy (4.5%), Greece (8.2%), Turkey (16.3% and 16.8% for the Southeastern Anatolia and Cukurova Regions, respectively) and Libya (28.4%) [5–9]. We also observed 18 other additional allelic variants in our haplotype dataset, including some further intermediate alleles: 1 haplotype with DYS385*12.2, 2 haplotypes with DYS392*10.2, 2 haplotypes with DYS458*21, 1 haplotype with DYS458*14.3, 6 haplotypes with DYS458*13, 2 haplotypes with DYS456*12, 1 haplotype with DYS635*17, 1 haplotype with DYS438*9.4, 1 haplotype with Y-GATA-H4*14, and 1 haplotype with DYS437*12. To our knowledge, while the intermediate allelic variant DYS438*9.4 has never been reported before, either in the literature or in databases such as YHRD [4] and STRBase [10], the intermediate allelic variant DYS458*14.3 has been reported only once in the STRBase. As pointed out in Gusmao et al. [3], intermediate alleles of similar type have already been detected at these two loci. Nevertheless, the DYS458*14.3 and DYS438*9.4 variants we have observed in our dataset were confirmed by double stranded DNA sequence analysis at the U.S. National Institute of Standards and Technology (NIST) (data not shown). Allelic variants may not only help better understand regional population genetics, but also help increase the discrimination power of DNA-based evidence. Haplotype frequencies were calculated using the Arlequin software suit v.3.5.1 [11]. Haplotype diversity (HD) was determined according to the Nei’s formula: HD = n/(n 1)(1 SPi2), where n is the population size and Pi is the frequency of the ith haplotype [12]. Haplotype diversity was calculated to be 0.9992, whereby the calculations excluded the diploid DYS385a/b loci altogether (Table S1). Gene diversity (GD) (equivalent to the power of discrimination) values for each locus were also calculated with the same equation, albeit using allele frequencies rather than haplotype frequencies (Table S2). Calculated average GD values were 0.6629 when the DYS385a/b was included as a diploid locus and 0.6429 when this locus was excluded all together. Pairwise genetic distance comparisons of the Turkish Cypriot Y-STR haplotype dataset with those from neighbouring populations [e.g., from Turkey (YA003668, n = 249, 17 loci for the Cukurova region; YA003727, n = 86, 17 loci for the Southeastern Anatolia region; YA003265, n = 113, 11 loci for the Central Anatolia region), Greece (YA003465, n = 191, 17 loci for the Northern Greeks), Israel/ Palestinian Authority area (YA003643, n = 155, 17 loci for Christian and Muslim Arabs), Egypt (YA003080, n = 83, 9 loci), and Italy (YA003721–26 & YA003744, n = 292, 17 loci)] and relatively distant populations [e.g., from Lithuania (YA003661, n = 194, 17 loci), Taiwan (YA003500, n = 208, 17 loci for the Paiwan population), and Australia (YA003697, n = 766, 17 loci for the Aboriginal population)] were carried out using the online tool at YHRD for the analysis of molecular variance (AMOVA) [4–8,13–18] (Table S3). The YHRD AMOVA tool uses the DYS389B allelic values obtained by subtracting the number of repeats at DYS389I from that of DYS389II, and excludes the DYS385a/b loci. Bonferroni correction was also applied to adjust for potential type I errors [19]. Finally,
K. Teralı et al. / Forensic Science International: Genetics 10 (2014) e1–e3
the SPSS Statistics software was used for MDS (multi-dimensional scaling) analysis in two dimensions using the genetic distance (Fst) values obtained during the AMOVA calculations (Figure S1) [20]. As summarized in Table S3, AMOVA results suggest that the calculated Fst values between the Turkish Cypriot population and those from the neighbouring populations ranged from 0.0064 to 0.0645, and those from the relatively distant populations ranged from 0.1203 to 0.4118. Based on the Fst values, the Turkish Cypriot population was closest to those populations from Turkey that are also the closest geographically, i.e. the Southeastern Anatolia (Fst = 0.0064) and the Cukurova (Fst = 0.0150) regions. After the Bonferroni correction, the P-values observed for the pairwise genetic comparisons between the Turkish Cypriot and the Southeastern Anatolia (P = 0.0776) and the Cukurova (P = 0.0002) populations suggested that the Turkish Cypriot population was most closely related to that from the Southeastern Anatolia first and then to that at Cukurova. Indeed, results from the MDS analysis confirmed that the Turkish Cypriot population was closest to that from the Southeastern Anatolia in both dimensions, and less so to those from Cukurova, Northern Greece and Egypt. On the MDS plot the Turkish Cypriots dissociated from the Israel/ Palestinian Authority area, Lithuania and Taiwan populations at least in one dimension or in both dimensions from the Central Anatolia, Italy and Australia populations. It must be noted that a major limitation during our AMOVA and subsequent MDS analyses was the relatively limited availability of comparable Y-STR datasets from around the Eastern Mediterranean region. To our knowledge, the Turkish Cypriot Y-STR haplotype dataset presented here constitutes the first of its kind from Cyprus in the literature. In this respect, it is expected to contribute to a better understanding of the population genetics of the Eastern Mediterranean basin. 7. Quality control All DNA extractions and subsequent Y-STR haplotyping were carried out at the Turkish Cypriot DNA Laboratory, whose proficiency has recently been certified through participation in the Y-STR Haplotyping Quality Assurance Exercise 2013 organized by the YHRD (www.yhrd.org) [4]. Data presented herein have also been submitted to the YHRD for further quality checks in advance of publication and received the following accession number: Cyprus [Turkish Cypriot] YA003850. 8. Other remarks This manuscript follows the guidelines for the publication of data requested by the journal [21]. Acknowledgements We gratefully acknowledge Mrs. Gulden Plumer Kucuk, the Turkish Cypriot Member of the Committee on Missing Persons in Cyprus (CMP), and all her staff, as well as all other volunteers who generously participated in this study. Financial support for this study has been provided by the CMP Turkish Cypriot Member Office (K.T. & C.G.) and the TRNC Presidency (C.G.). We are also very grateful to Ms. Carollyn R. Hill and Dr. Peter Vallone, both at NIST,
e3
for the characterization of the DYS458*14.3 and DYS438*9.4 allelic variants by sequencing. The primary purpose of this population study is to contribute to the ongoing CMP Project on the ‘‘Exhumation, Identification and Return of Remains of Missing Persons’’, as well as to provide foundations for the forensic genetic services in North Cyprus. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.fsigen.2014.01.003. References [1] The Earliest Prehistory of Cyprus: From Colonization to Exploitation, S. Swinny (Ed.), Cyprus American Archaeological Research Institute Monograph Series, Vol. 2, American Schools of Oriental Research Publications, Boston, 2001. [2] Applied Biosystems, AmpFlSTR1 Yfiler TM PCR Amplification Kit User’s Manual, Foster City, CA, 2004. [3] L. Gusmao, J.M. Butler, A. Carracedo, P. Gill, M. Kayser, W.R. Mayr, N. Morling, M. Prinz, L. Roewer, C. Tyler-Smith, P.M. Schneider, DNA Commission of the International Society of Forensic Genetics (ISFG): an update of the recommendations on the use of Y-STRs in forensic analysis, Forensic Sci. Int. 157 (2006) 187–197. [4] S. Willuweit, L. Roewer, Y chromosome haplotype reference database (YHRD): update, Forensic Sci. Int. Genet. (2007) 83–87. [5] F. Brisighelli, A. Blanco-Verea, I. Boschi, P. Garaghnani, V.L. Pascali, A. Carracedo, C. Capelli, A. Salas, Patterns of Y-STR variation in Italy, Forensic Sci. Int. Genet. 6 (2012) 834–839. [6] L. Kovatsi, J.L. Saunier, J.A. Irwin, Population genetics of Y-chromosome STRS in a population of Northern Greeks, Forensic Sci. Int. Genet. 4 (2009) e21–e22. [7] A. Serin, H. Canan, B. Alper, Y. Sertdemir, Haplotype frequencies of 17 Y-chromosomal short tandem repeat loci from the Cukurova region of Turkey, Croat. Med. J. 52 (2011) 703–708. [8] F. Ozbas-Gerceker, N. Bozman, A. Arslan, A. Serin, Population data for 17 Y-STRs in samples from Southeastern Anatolia Region of Turkey, Int. J. Hum. Genet. 13 (2013) 105–111. [9] S. Triki-Fendri, P. Sanchez-Diz, D. Rey-Gonzales, I. Ayadi, S. Alfadhli, A. Rebai, A. Carracedo, Population genetics of 17-Y-STR markers in West Libya (Tripoli region), Forensic Sci. Int. Genet. 7 (2013) e59–e61. [10] C.M. Ruitberg, D.J. Reeder, J.M. Butler, STRBase: a short tandem repeat DNA database for the human identity testing community, Nucleic Acids Res. 29 (2001) 320–322. [11] L. Excoffier, H.E. Lischer, Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows, Mol. Ecol. Resour. 10 (3) (2010) 564–567. [12] M. Nei, Molecular Evolutionary Genetics, Columbia University Press, New York, 1987, pp. 176–179. [13] A.H. Cakir, A. Celebioglu, E. Yardimci, Y-STR haplotypes in Central Anatolia region of Turkey, Forensic Sci. Int. 144 (2004) 59–64. [14] A.T. Fernandes, R. Goncalves, S. Gomes, D. Filon, A. Nebel, M. Faerman, A. Brehm, Y-chromosomal STRs in two populations from Israel and the Palestinian Authority Area: Christian and Muslim Arabs, Forensic Sci. Int. Genet. 5 (2011) 561–562. [15] F. Manni, P. Leonardi, A. Barakat, H. Rouba, E. Heyer, M. Klintschar, K. Mcelreavey, L. Quintana-Murci, Y-chromosome analysis in Egypt suggests a genetic regional continuity in Northeastern Africa, Hum. Biol. 74 (2002) 645–658. [16] M. Caplinskiene, A. Pauliukevicius, J. Jankauskiene, D. Bunokiene, J. Kukiene, K. Savanevskyte, A. Jureniene, Autosomal and Y-STR mutations in Lithuanian population, Forensic Sci. Int. Genet. Suppl. 1 (2008) 237–238. [17] F.-C. Wu, C.-W.C.-E.P. Ho, K.-Y. Hu, S. Willuweit, L. Roewer, D.H. Liu, Y-chromosomal STRs in the Taiwanese Paiwan population, Int. J. Legal Med. 125 (2011) 39– 43. [18] D.A. Taylor, J.M. Henry, Haplotype data for 16 Y-chromosome STR loci in Aboriginal and Caucasian populations in South Australia, Forensic Sci. Int. Genet. 6 (2012) e187–e188. [19] Y. Hochberg, A sharper Bonferroni procedure for multiple tests of significance, Biometrika 75 (4) (1988) 800–802. [20] SPSS Statistics for Windows, version 21, SPSS Inc., Chicago, IL. [21] A. Carrracedo, J.M. Butler, L. Gusmao, A. Linacre, W. Parson, L. Roewer, P.M. Schneider, New guidelines for the publication of genetic population data, Forensic Sci. Int. Genet. 7 (2013) 217–220.
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Forensic Population Genetics - Short Communication
Population genetics of 17 Y-STR markers in Turkish Cypriots from Cyprus§ K. Teralı a, T. Zorlu b, O. Bulbul b, C. Gurkan a,* a b
Turkish Cypriot DNA Laboratory, Committee on Missing Persons in Cyprus Turkish Cypriot Member Office, Nicosia, North Cyprus Institute of Forensic Science, Istanbul University, Istanbul, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 October 2013 Received in revised form 7 January 2014 Accepted 8 January 2014
We analyzed seventeen Y-chromosomal short tandem repeats (STRs) [DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y-GATA-H4, DYS437, DYS438, and DYS448] in 253 unrelated, male individuals from the Turkish Cypriot population of the Eastern Mediterranean island of Cyprus. While 206 out of the 253 haplotypes present in the dataset were unique, there are also 22 haplotypes that were observed in two individuals each, and 1 haplotype that was observed in three individuals. While no locus duplications or null alleles were observed in our dataset, we have detected 43 allelic variants in total, the majority of which (25 out of 253 haplotypes or 9.88%) comprised of .2 intermediate variants at the DYS458 locus (alleles 16.2, 17.2, 18.2, 19.2, and 20.2). For the 229 different haplotypes observed in the Turkish Cypriot dataset, the calculated discrimination capacity (DC) was 0.9051 and the haplotype diversity (HD) was 0.9992. The calculated average gene diversity (GD) values ranged from 0.3828 to 0.9631 for the DYS392 and DYS385a/b loci, respectively. Pairwise genetic distance comparisons of the Turkish Cypriot Y-STR dataset with those from the neighbouring (Turkey, Greece, Israel/Palestinian Authority area, Egypt and Italy) and relatively distant (Lithuania, Taiwan and Australia) countries through the use of analysis of molecular variance (AMOVA) and multi-dimensional scaling (MDS) analyses confirmed that our data do not deviate significantly from the typical core haplotypes of the Eastern Mediterranean region. The Turkish Cypriot Y-STR haplotype dataset will find an immediate use in the Committee on Missing Persons in Cyprus Project on the ‘‘Exhumation, Identification and Return of Remains of Missing Persons’’ and it is also expected to contribute to the establishment of forensic genetic services in North Cyprus. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Y-filer Y-STR Short tandem repeats (STRs) Cyprus Turkish Cypriot
1. Population Cyprus is the third largest island in the Mediterranean Sea situated 75 km to the South of Turkey, 200 km to the North West of Israel/Palestinian Authority area, 380 km to the North of Egypt, and 800 km to the South East of the Greek mainland. The earliest confirmed site of human activity on the island dates back to around 12,000 years before present [1]. Strategically situated at the crossroads of major civilizations and historical population movements, Cyprus subsequently received waves of ancient Greek settlements and Phoenician colonizations, ruled by the Assyrian, Persian, Hellenistic, Roman and Byzantine Empires, the latter of which was also intermitted with Arab conquests and an ArabByzantine condominium, taken over by the Crusaders/Lusignans/
§ Access to data is available upon request from the corresponding author ([email protected]). * Corresponding author. Tel.: +90 542 8584783; fax: +90 392 2238856. E-mail address: [email protected] (C. Gurkan).
1872-4973/$ – see front matter ß 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigen.2014.01.003
Venetians, conquered by the Ottoman Empire and finally occupied by the British Empire prior to her independence in 1960. Today, there are two major ethnic groups on the island, namely the Turkish Cypriots who speak Turkish and are Muslims and the Greek Cypriots who speak Greek and are mostly Orthodox Christians. There are also other constitutionally recognized ethnic groups that include Maronites, Armenians, and Latins. The subject of this study, the Turkish Cypriot population, resided all over the island in villages or cities wherein the populace comprised of either mixed ethnic background or exclusively Turkish Cypriot up until 1963/1974, but has largely relocated/concentrated in the Turkish Cypriot administrated North Cyprus since 1974. 2. Sampling Blood or buccal swab samples were collected along with signed informed consents from 253 unrelated, male individuals from the Turkish Cypriot population currently residing in North Cyprus. The participants also filled out a brief survey on their ethnic origin,
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K. Teralı et al. / Forensic Science International: Genetics 10 (2014) e1–e3
which included questions on their birthplace and that of their parents, as well as any other additional information that they may have with respect to their paternal pedigree. For the purposes of this study, we aimed to select each participant among volunteers who were born in Cyprus and/or currently residing in North Cyprus, but also born to Turkish Cypriot parents, whereby for the large majority of cases both father and mother were born in Cyprus, and if not, at least the father was born in Cyprus. Considering the relatively recent mass population movements of the Turkish Cypriots across the island over the last fifty years or so as described above, the traditional paternal birthplace of the volunteers were assumed as the origin of each given Y-STR haplotype (Ht) observed. 3. DNA extraction Genomic DNA extraction was carried out using the PureLinkTM Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. 4. PCR amplification PCR for 17 Y-STR loci [DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y-GATA-H4, DYS437, DYS438, and DYS448] was performed in a multiplex reaction using the AmpFlSTR1 Y-filerTM PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA). The total amplification reaction volume was 25 ml, which contained 9.2 ml of reaction mix, 5.0 ml of primer set, 0.8 ml of AmpliTaq Gold DNA Polymerase and 10 ml of genomic DNA (approximately 0.5– 1 ng ml 1). Amplifications were performed as described in the instruction manual of the AmpFlSTR1 Y-filer TM PCR Amplification Kit and using the Applied Biosystems 2720 Thermal Cycler (Applied Biosystems) [2]. 5. Electrophoresis and typing Separation and detection of the 17 polymorphic Y-chromosome-specific PCR amplicons were performed using the ABI 3130 Genetic Analyzer (Applied Biosystems). Each sample was prepared by adding 1.5 ml of PCR product to a mixture of 8.4 ml of Hi-DiTM formamide and 0.6 ml of GeneScanTM-500 LIZTM Internal Size Standard (both Applied Biosystems). Samples were analyzed using GeneMapper v4.0 (Applied Biosystems). Alleles were assigned according to the current International Society of Forensic Genetics (ISFG) guidelines for forensic Y-STR analysis [3]. 6. Data analyses Among the 253 haplotypes observed in the Turkish Cypriot YSTR dataset, 22 haplotypes were each detected in two individuals and 1 haplotype was detected in three individuals (Table S1). Notably, 2 out of the 3 individuals who share the same haplotype that was observed in triplicate (Ht 035), as well as 7 out of the 22 pairs of individuals who share the same haplotype in each pair (Hts 066, 070, 079, 087, 142, 166, and 193), were also found to share the same paternal geographic origins (i.e., small, isolated villages) in each pair, albeit without any evidence toward close paternal relatedness. In other words, a total of 8 pairs of seemingly unrelated individuals were found to share not only the same haplotype in each pair, but also the same paternal geographic origin. These observations perhaps could be attributed to a past tendency toward patrilocal residence patterns in isolated subpopulations of Turkish Cypriots. The Turkish Cypriot Y-STR dataset has 206 unique haplotypes (UH) (81.42%; number of unique haplotypes observed/total number of samples analyzed 100) and
229 different haplotypes, thus yielding a very high discrimination capacity (DC) (0.9051, total number of different haplotypes observed/total number of samples analyzed). The most common haplotype observed in the dataset is found at a frequency of 1.19% (Ht 035, observed in triplicate), which has so far never been reported in the YHRD database (as of 22 August 2013) [4]. While we did not observe any locus duplications or null alleles in the 253 samples analyzed, based on the observed fragment sizes, we observed 43 allelic variants in total. 25 of the allelic variants (9.88% of all haplotypes) we observed were .2 intermediate alleles at the DYS458 locus, namely 16.2, 17.2, 18.2, 19.2 and 20.2. Presence of this relatively high percentage of DYS458*.2 variants (9.88%) in the Turkish Cypriot population compares well with those from geographically and/or historically related countries, such as Italy (4.5%), Greece (8.2%), Turkey (16.3% and 16.8% for the Southeastern Anatolia and Cukurova Regions, respectively) and Libya (28.4%) [5–9]. We also observed 18 other additional allelic variants in our haplotype dataset, including some further intermediate alleles: 1 haplotype with DYS385*12.2, 2 haplotypes with DYS392*10.2, 2 haplotypes with DYS458*21, 1 haplotype with DYS458*14.3, 6 haplotypes with DYS458*13, 2 haplotypes with DYS456*12, 1 haplotype with DYS635*17, 1 haplotype with DYS438*9.4, 1 haplotype with Y-GATA-H4*14, and 1 haplotype with DYS437*12. To our knowledge, while the intermediate allelic variant DYS438*9.4 has never been reported before, either in the literature or in databases such as YHRD [4] and STRBase [10], the intermediate allelic variant DYS458*14.3 has been reported only once in the STRBase. As pointed out in Gusmao et al. [3], intermediate alleles of similar type have already been detected at these two loci. Nevertheless, the DYS458*14.3 and DYS438*9.4 variants we have observed in our dataset were confirmed by double stranded DNA sequence analysis at the U.S. National Institute of Standards and Technology (NIST) (data not shown). Allelic variants may not only help better understand regional population genetics, but also help increase the discrimination power of DNA-based evidence. Haplotype frequencies were calculated using the Arlequin software suit v.3.5.1 [11]. Haplotype diversity (HD) was determined according to the Nei’s formula: HD = n/(n 1)(1 SPi2), where n is the population size and Pi is the frequency of the ith haplotype [12]. Haplotype diversity was calculated to be 0.9992, whereby the calculations excluded the diploid DYS385a/b loci altogether (Table S1). Gene diversity (GD) (equivalent to the power of discrimination) values for each locus were also calculated with the same equation, albeit using allele frequencies rather than haplotype frequencies (Table S2). Calculated average GD values were 0.6629 when the DYS385a/b was included as a diploid locus and 0.6429 when this locus was excluded all together. Pairwise genetic distance comparisons of the Turkish Cypriot Y-STR haplotype dataset with those from neighbouring populations [e.g., from Turkey (YA003668, n = 249, 17 loci for the Cukurova region; YA003727, n = 86, 17 loci for the Southeastern Anatolia region; YA003265, n = 113, 11 loci for the Central Anatolia region), Greece (YA003465, n = 191, 17 loci for the Northern Greeks), Israel/ Palestinian Authority area (YA003643, n = 155, 17 loci for Christian and Muslim Arabs), Egypt (YA003080, n = 83, 9 loci), and Italy (YA003721–26 & YA003744, n = 292, 17 loci)] and relatively distant populations [e.g., from Lithuania (YA003661, n = 194, 17 loci), Taiwan (YA003500, n = 208, 17 loci for the Paiwan population), and Australia (YA003697, n = 766, 17 loci for the Aboriginal population)] were carried out using the online tool at YHRD for the analysis of molecular variance (AMOVA) [4–8,13–18] (Table S3). The YHRD AMOVA tool uses the DYS389B allelic values obtained by subtracting the number of repeats at DYS389I from that of DYS389II, and excludes the DYS385a/b loci. Bonferroni correction was also applied to adjust for potential type I errors [19]. Finally,
K. Teralı et al. / Forensic Science International: Genetics 10 (2014) e1–e3
the SPSS Statistics software was used for MDS (multi-dimensional scaling) analysis in two dimensions using the genetic distance (Fst) values obtained during the AMOVA calculations (Figure S1) [20]. As summarized in Table S3, AMOVA results suggest that the calculated Fst values between the Turkish Cypriot population and those from the neighbouring populations ranged from 0.0064 to 0.0645, and those from the relatively distant populations ranged from 0.1203 to 0.4118. Based on the Fst values, the Turkish Cypriot population was closest to those populations from Turkey that are also the closest geographically, i.e. the Southeastern Anatolia (Fst = 0.0064) and the Cukurova (Fst = 0.0150) regions. After the Bonferroni correction, the P-values observed for the pairwise genetic comparisons between the Turkish Cypriot and the Southeastern Anatolia (P = 0.0776) and the Cukurova (P = 0.0002) populations suggested that the Turkish Cypriot population was most closely related to that from the Southeastern Anatolia first and then to that at Cukurova. Indeed, results from the MDS analysis confirmed that the Turkish Cypriot population was closest to that from the Southeastern Anatolia in both dimensions, and less so to those from Cukurova, Northern Greece and Egypt. On the MDS plot the Turkish Cypriots dissociated from the Israel/ Palestinian Authority area, Lithuania and Taiwan populations at least in one dimension or in both dimensions from the Central Anatolia, Italy and Australia populations. It must be noted that a major limitation during our AMOVA and subsequent MDS analyses was the relatively limited availability of comparable Y-STR datasets from around the Eastern Mediterranean region. To our knowledge, the Turkish Cypriot Y-STR haplotype dataset presented here constitutes the first of its kind from Cyprus in the literature. In this respect, it is expected to contribute to a better understanding of the population genetics of the Eastern Mediterranean basin. 7. Quality control All DNA extractions and subsequent Y-STR haplotyping were carried out at the Turkish Cypriot DNA Laboratory, whose proficiency has recently been certified through participation in the Y-STR Haplotyping Quality Assurance Exercise 2013 organized by the YHRD (www.yhrd.org) [4]. Data presented herein have also been submitted to the YHRD for further quality checks in advance of publication and received the following accession number: Cyprus [Turkish Cypriot] YA003850. 8. Other remarks This manuscript follows the guidelines for the publication of data requested by the journal [21]. Acknowledgements We gratefully acknowledge Mrs. Gulden Plumer Kucuk, the Turkish Cypriot Member of the Committee on Missing Persons in Cyprus (CMP), and all her staff, as well as all other volunteers who generously participated in this study. Financial support for this study has been provided by the CMP Turkish Cypriot Member Office (K.T. & C.G.) and the TRNC Presidency (C.G.). We are also very grateful to Ms. Carollyn R. Hill and Dr. Peter Vallone, both at NIST,
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for the characterization of the DYS458*14.3 and DYS438*9.4 allelic variants by sequencing. The primary purpose of this population study is to contribute to the ongoing CMP Project on the ‘‘Exhumation, Identification and Return of Remains of Missing Persons’’, as well as to provide foundations for the forensic genetic services in North Cyprus. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.fsigen.2014.01.003. References [1] The Earliest Prehistory of Cyprus: From Colonization to Exploitation, S. Swinny (Ed.), Cyprus American Archaeological Research Institute Monograph Series, Vol. 2, American Schools of Oriental Research Publications, Boston, 2001. [2] Applied Biosystems, AmpFlSTR1 Yfiler TM PCR Amplification Kit User’s Manual, Foster City, CA, 2004. [3] L. Gusmao, J.M. Butler, A. Carracedo, P. Gill, M. Kayser, W.R. Mayr, N. Morling, M. Prinz, L. Roewer, C. Tyler-Smith, P.M. Schneider, DNA Commission of the International Society of Forensic Genetics (ISFG): an update of the recommendations on the use of Y-STRs in forensic analysis, Forensic Sci. Int. 157 (2006) 187–197. [4] S. Willuweit, L. Roewer, Y chromosome haplotype reference database (YHRD): update, Forensic Sci. Int. Genet. (2007) 83–87. [5] F. Brisighelli, A. Blanco-Verea, I. Boschi, P. Garaghnani, V.L. Pascali, A. Carracedo, C. Capelli, A. Salas, Patterns of Y-STR variation in Italy, Forensic Sci. Int. Genet. 6 (2012) 834–839. [6] L. Kovatsi, J.L. Saunier, J.A. Irwin, Population genetics of Y-chromosome STRS in a population of Northern Greeks, Forensic Sci. Int. Genet. 4 (2009) e21–e22. [7] A. Serin, H. Canan, B. Alper, Y. Sertdemir, Haplotype frequencies of 17 Y-chromosomal short tandem repeat loci from the Cukurova region of Turkey, Croat. Med. J. 52 (2011) 703–708. [8] F. Ozbas-Gerceker, N. Bozman, A. Arslan, A. Serin, Population data for 17 Y-STRs in samples from Southeastern Anatolia Region of Turkey, Int. J. Hum. Genet. 13 (2013) 105–111. [9] S. Triki-Fendri, P. Sanchez-Diz, D. Rey-Gonzales, I. Ayadi, S. Alfadhli, A. Rebai, A. Carracedo, Population genetics of 17-Y-STR markers in West Libya (Tripoli region), Forensic Sci. Int. Genet. 7 (2013) e59–e61. [10] C.M. Ruitberg, D.J. Reeder, J.M. Butler, STRBase: a short tandem repeat DNA database for the human identity testing community, Nucleic Acids Res. 29 (2001) 320–322. [11] L. Excoffier, H.E. Lischer, Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows, Mol. Ecol. Resour. 10 (3) (2010) 564–567. [12] M. Nei, Molecular Evolutionary Genetics, Columbia University Press, New York, 1987, pp. 176–179. [13] A.H. Cakir, A. Celebioglu, E. Yardimci, Y-STR haplotypes in Central Anatolia region of Turkey, Forensic Sci. Int. 144 (2004) 59–64. [14] A.T. Fernandes, R. Goncalves, S. Gomes, D. Filon, A. Nebel, M. Faerman, A. Brehm, Y-chromosomal STRs in two populations from Israel and the Palestinian Authority Area: Christian and Muslim Arabs, Forensic Sci. Int. Genet. 5 (2011) 561–562. [15] F. Manni, P. Leonardi, A. Barakat, H. Rouba, E. Heyer, M. Klintschar, K. Mcelreavey, L. Quintana-Murci, Y-chromosome analysis in Egypt suggests a genetic regional continuity in Northeastern Africa, Hum. Biol. 74 (2002) 645–658. [16] M. Caplinskiene, A. Pauliukevicius, J. Jankauskiene, D. Bunokiene, J. Kukiene, K. Savanevskyte, A. Jureniene, Autosomal and Y-STR mutations in Lithuanian population, Forensic Sci. Int. Genet. Suppl. 1 (2008) 237–238. [17] F.-C. Wu, C.-W.C.-E.P. Ho, K.-Y. Hu, S. Willuweit, L. Roewer, D.H. Liu, Y-chromosomal STRs in the Taiwanese Paiwan population, Int. J. Legal Med. 125 (2011) 39– 43. [18] D.A. Taylor, J.M. Henry, Haplotype data for 16 Y-chromosome STR loci in Aboriginal and Caucasian populations in South Australia, Forensic Sci. Int. Genet. 6 (2012) e187–e188. [19] Y. Hochberg, A sharper Bonferroni procedure for multiple tests of significance, Biometrika 75 (4) (1988) 800–802. [20] SPSS Statistics for Windows, version 21, SPSS Inc., Chicago, IL. [21] A. Carrracedo, J.M. Butler, L. Gusmao, A. Linacre, W. Parson, L. Roewer, P.M. Schneider, New guidelines for the publication of genetic population data, Forensic Sci. Int. Genet. 7 (2013) 217–220.