Forensic Science International: Genetics 7 (2013) e56–e58

Forensic Population Genetics – Short communication

Genetic population study of 11 Y chromosome STR loci in Greece

Panayotis Katsaloulis b, Konstantina Tsekoura a, Maria Vouropoulou a, Penelope Miniati a,*a Department of Biological Material Analysis, Division of Forensic Science, Hellenic Police, Sevastoupoleos 14, 11526 Athens, Greeceb Institute of Physical Chemistry, National Center for Scientific Research ‘‘Demokritos’’, 15310 Athens, Greece

A R T I C L E I N F O

Article history:

Received 9 March 2012

Received in revised form 27 November 2012

Accepted 6 February 2013

Keywords:

Y chromosome

STR

PowerPlex

Allele frequencies

Greek population

A B S T R A C T

Statistical properties of eleven Y chromosome Short Tandem Repeat (STR) markers were analyzed

(DYS391, DYS389I, DYS439, DYS389II, DYS438, DYS437, DYS19, DYS392, DYS393, DYS390 and DYS385)

in a Greek population sample. The 200 subjects where distributed across Greece, from various

Peripheries. 182 distinct haplotypes were found. To validate our results gene diversity has been

calculated for the whole population, as well as for each locus individually. Genetic distance has been

estimated between this population and Albanian, Egyptian, Italian and Turkish populations. The results

indicate that all Y loci are useful for forensic sciences.

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1. Population

Blood samples of 200 healthy unrelated subjects were obtained.The regional distribution of the subjects is across Greece from thePeripheries Western Greece, Attica, Peloponnese, East Macedoniaand Thrace, North Aegean, Western Macedonia, Hepirus, IonianIslands, Central Macedonia, Crete and Southern Aegean, StereaEllada and Thessaly. The number of samples per Periphery ispresented in Fig. 1. Due to increased internal immigration of thepopulation during the last decades in Greece [1], the birth place ofthe father was taken into account, when assigning the Periphery ofeach subject.

The samples were processed for DNA extraction and analysis inthe laboratory of the Department of Biological Material Analysis,Hellenic Police. No connections or relationship between thesubjects were known. The procedure we followed is in accordancewith the proposed methodology on Y chromosome STR analysis[2–4], similar to the methodology presented in [5–11].

2. Extraction

The standard protocol of human and animal tissue wasfollowed, to extract genomic DNA [12–14]. The kit used was‘‘NucleoSpin Tissue Kit of MACHEREY-NAGEL’’. Extracted DNA was

* Corresponding author. Tel.: þ30 210 5103412; fax: þ30 210 5103408.

E-mail addresses: pkatsaloulis@chem.demokritos.gr (P. Katsaloulis),

dna@astynomia.gr (P. Miniati).

1872-4973/$ – see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.fsigen.2013.02.001

quantified by Real-Time Polymerase Chain Reaction (RT-PCR),using Quantifiler Kit of Applied Biosystems (ABI).

3. PCR

Eleven loci situated in the Y chromosome were examined. Allthese areas represent well-characterized loci, common in forensiccases and population studies.

The PCR was performed according to PowerPlex Y SystemTechnical Manual (Promega Corporation, Madison, WI, USA) using1 ng of template DNA [15]. For the amplification of the samples therespective protocol was followed, using a GeneAmp PCR System9700 thermal cycler of Applied Biosystems. The 9948 male DNAfrom Promega was used as positive control (PC) in the PCRprocedure.

4. Typing

The amplified fragments were detected with the AppliedBiosystems Prism 3100 Genetic Analyzer. The electropherogramswere analyzed by GeneMapper ID Software version 3.1. Automaticallele calling was applied, electropherograms were visuallyinspected and allele calls were verified.

5. Quality control

The laboratory undergoes internal standard testing. It alsoparticipates in GEDNAP blind trials for the aforementioned Y lociand is awarded with the respective proficiencies.

Fig. 1. Distribution of samples across the Greek Peripheries. The names of the

Peripheries are described in Table 1.

Table 1Haplotype diversity values between Greek Peripheries. N is the number of subjects

in every Periphery. The number of distinct haplotypes per Periphery is indicated in

h. The total number of distinct haplotypes (182) does not coincide with the sum of h

in this table (197), since subjects from different Peripheries share the same

haplotype. The Peripheries abbreviations are also presented.

Peripheries Abbreviation N h

Western Greece DE 31 31

Attica AT 25 25

Peloponnese PE 19 18

East Macedonia and Thrace AMTH 21 21

North Aegean BA 11 11

Western Macedonia DM 10 10

Hepirus HP 18 18

Ionian Islands IN 7 7

Central Macedonia KM 25 23

Crete and Southern Aegean KRNA 6 6

Sterea Ellada SE 17 17

Thessaly THE 10 10

Table 2Calculated pairwise FST and p-values distances between (a) the population

presented here (Greece), (b) population from Northern Greece [5] and (c) from

Continental Greece and the islands of Crete and Chios [7].

Population Greece N. Greece CG/C/C

Greece – 0.1616 0.8631

N. Greece 0.0003 – 0.1386

CG/C/C �0.0004 0.0002 –

P. Katsaloulis et al. / Forensic Science International: Genetics 7 (2013) e56–e58 e57

The population fulfills the recommendations of the DNACommission of the International Society of Forensic Genetics[16] and has been accepted in the Y chromosome HaplotypeReference Database with Accession Number YA003647.

6. Analysis of data

Haplotype (gene) diversity values h were calculated using theformula [17]

h ¼ N

N � 11 �

Xp2

i

� �(1)

where N is the population size (namely 200 subjects) and pi is thefrequency of the ith haplotype. Standard error SE was alsocalculated according to the equation

SE ¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

2

Pp3

i �P

p2i

� �2n o

N

8<:

9=;

vuuut (2)

In this work we have calculated the gene diversity for the wholepopulation, as well as for each locus individually. The statisticalanalysis has been performed with Microsoft Office XP Excel.

7. Results and discussion

A total of 200 subjects from Greece were investigated in thisstudy. Analysis revealed 182 distinct haplotypes, with haplotypediversity h of the whole population h = 0.9986 with SE = 0.00038.Only 12 out of 182 haplotypes, a mere 6.6%, were found more thanonce in this population. We can note also that only one haplotypewas found five times, while all others appear three times or less. Inour data the diversity of each locus is usually between four andseven alleles. Four allele variations appear in loci DYS389-I andDYS437, both of them having a single representation of allele 15 inthe former, and allele 17 in the latter. Five alleles appear in lociDYS391, DYS392 and DYS438. Six alleles appear in loci DYS19,DYS389-II, DYS390 and DYS393, while seven alleles in locusDYS439. Locus DYS385 was found with 13 alleles, as we take into

account that the gene DYS385 is duplicated and consists of twocopies (DYS385a and DYS385b) [18–20].

For every locus the gene diversity has also been calculated andpresented. The locus with the highest diversity is locus DYS385with values h = 0.9537 and SE � 0.00396. This is expected since thislocus has the highest number of allelic variation and also correspondsto a duplicated gene. Locus DYS19 has values of h = 0.7671 andSE � 0.00648, followed by DYS389-II (h = 0.7471 and SE � 0.01039)and DYS439 (h = 0.718 and SE � 0.01187). All other loci have genediversity with value h < 0.7. The locus with the smallest genediversity is DYS392 with values h = 0.3709 and SE � 0.02689.

The haplotypes found per Periphery is presented in Table 1. Theobserved discrepancy between the 182 distinct haplotypes in thewhole data-set and the sum of distinct haplotypes per Periphery(197) is due to the fact that subjects from different Peripheriesshare the same haplotype.

In most of the cases, no haplotype duplicity exists in ourspecimens, or if it exists, the duplicate haplotypes are very few. Forthis reason, the calculation of haplotype gene diversity is of nopractical value.

Genetic distance between neighbor populations has beenestimated with the Analysis of Molecular Variance tool (AMOVA[23], YHDR database [24]). In Table 2 we present the calculatedpairwise FST and p-values distances between the populationpresented here and other Greek populations; a population fromNorthern Greece [5] and a population from Continental Greece andthe islands of Crete and Chios [7] have been taken into account. Theparameters chosen are threshold of TFST

¼ 0:1, minimal size ofcluster Smin = 10 and 10.000 permutations. Similar statistics havebeen performed between this Greek population and an Albanianpopulation set of 8 population samples with 559 haplotypes, anEgyptian population set of 6 population samples with 291haplotypes, an Italian population set of 40 population sampleswith 3555 haplotypes, a Spanish population set of 20 populationsamples with 2692 haplotypes and a Turkish population set of 13

Table 3Calculated pairwise FST and p-values distances between Albanian, Egyptian, Italian,

Spanish, Turkish populations and Greek population with 10,000 permutations.

Population Albania Egypt Italy Spain Turkey Greece

Albania – 0.0000 0.0000 0.0000 0.0000 0.0000

Egypt 0.0048 – 0.0014 0.0000 0.0000 0.0005

Italy 0.0046 0.0005 – 0.0000 0.0000 0.0001

Spain 0.0064 0.0021 0.0010 – 0.0000 0.0000

Turkey 0.0053 0.0009 0.0011 0.0027 – 0.0000

Greece 0.0036 0.0007 0.0008 0.0024 0.0013 –

Fig. 2. Nonlinear Mapping For Data Structure Analysis for the Greek, Albanian,

Egyptian, Italian, Spanish and Turkish population.

P. Katsaloulis et al. / Forensic Science International: Genetics 7 (2013) e56–e58e58

population samples with 1302 haplotypes, presented in Table 3. InFig. 2 the Mapping for Data Structure Analysis (MDS plot) usingSammon mapping [21,22] is presented. The genetic distancebetween the Greek and the Italian and Egyptian population issmaller compared with the Turkish and Spanish populations, withthe Albanian population to have the largest distance between thesepopulations.

The gene diversity of the Y chromosome related STRs has beenfound to be statistically important, in the presented population.This indicates that the usage of Y specific STRs are useful as aforensic evidence for the Greek population.

8. Other remarks

This paper follows the guidelines for publication of populationdata requested by the journal [25].

Appendix A. Supplementary Data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.fsigen.2013.02.001.

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