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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 manufacturers 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 per-

formed 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 contained9.2 ml of reaction mix,5.0 ml of primer set, 0.8 ml of AmpliTaq GoldDNA Polymerase and 10 ml of genomic DNA (approximately 0.51 ngml1). Amplifications were performed as described in theinstruction manual of the AmpFlSTR1 Y-filerTM 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-chromo-some-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

Y-

STR 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 samehaplotype in eachpair (Hts

066, 070, 079, 087, 142, 166, and193),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 sub-

populations of Turkish Cypriots. The Turkish Cypriot Y-STR dataset

has

206

unique

haplotypes

(UH)

(81.42%;

number

of

unique

haplotypesobserved/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

inthe253 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%) [59]. 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

withDYS437*12. To our knowledge, while the intermediate allelicvariant 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 deter-mined according to the Neis formula: HD = n/(n 1)(1 SPi

2),

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

Anatoliaregion;

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

(YA00372126 &

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 popula-

tion), 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)

[48,1318]

(Table

S3).

TheYHRDAMOVA tool uses theDYS389Ballelic values obtainedby

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. Teral et al./ Forensic Science International: Genetics 10 (2014) e1e3e2

8/9/2019 1-s2.0-S1872497314000040-main

3/3

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

calculatedFstvalues between the Turkish Cypriot population andthose from the neighbouring populations ranged from 0.0064 to

0.0645, and those from the relatively distant populations ranged

from 0.1203 to0.4118. Based on theFstvalues, the Turkish Cypriotpopulation 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 theBonferroni 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

Anatoliafirst and then to that atCukurova. 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 amajor limitationduring our AMOVA and subsequentMDS analyses

was the relatively limited availability of comparable Y-STR

datasets from around the Eastern Mediterranean region. To our

knowledge, the TurkishCypriotY-STRhaplotypedatasetpresented

hereconstitutes 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 organizedby 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 inCyprus (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,

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 .

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