Genetic population study of 11 Y chromosome STR loci in Greece

Genetic population study of 11 Y chromosome STR loci in Greece

Forensic Science International: Genetics 7 (2013) e56–e58 Contents lists available at SciVerse ScienceDirect Forensic Science International: Genetic...

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

Contents lists available at SciVerse ScienceDirect

Forensic Science International: Genetics journal homepage:

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 b

Department of Biological Material Analysis, Division of Forensic Science, Hellenic Police, Sevastoupoleos 14, 11526 Athens, Greece Institute of Physical Chemistry, National Center for Scientific Research ‘‘Demokritos’’, 15310 Athens, Greece



Article history: Received 9 March 2012 Received in revised form 27 November 2012 Accepted 6 February 2013

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. ß 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Y chromosome STR PowerPlex Allele frequencies Greek population

1. Population Blood samples of 200 healthy unrelated subjects were obtained. The regional distribution of the subjects is across Greece from the Peripheries Western Greece, Attica, Peloponnese, East Macedonia and Thrace, North Aegean, Western Macedonia, Hepirus, Ionian Islands, Central Macedonia, Crete and Southern Aegean, Sterea Ellada and Thessaly. The number of samples per Periphery is presented in Fig. 1. Due to increased internal immigration of the population during the last decades in Greece [1], the birth place of the father was taken into account, when assigning the Periphery of each subject. The samples were processed for DNA extraction and analysis in the laboratory of the Department of Biological Material Analysis, Hellenic Police. No connections or relationship between the subjects were known. The procedure we followed is in accordance with 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 was followed, to extract genomic DNA [12–14]. The kit used was ‘‘NucleoSpin Tissue Kit of MACHEREY-NAGEL’’. Extracted DNA was

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. All these areas represent well-characterized loci, common in forensic cases and population studies. The PCR was performed according to PowerPlex Y System Technical Manual (Promega Corporation, Madison, WI, USA) using 1 ng of template DNA [15]. For the amplification of the samples the respective protocol was followed, using a GeneAmp PCR System 9700 thermal cycler of Applied Biosystems. The 9948 male DNA from Promega was used as positive control (PC) in the PCR procedure. 4. Typing The amplified fragments were detected with the Applied Biosystems Prism 3100 Genetic Analyzer. The electropherograms were analyzed by GeneMapper ID Software version 3.1. Automatic allele calling was applied, electropherograms were visually inspected and allele calls were verified. 5. Quality control

* Corresponding author. Tel.: þ30 210 5103412; fax: þ30 210 5103408. E-mail addresses: [email protected] (P. Katsaloulis), [email protected] (P. Miniati). 1872-4973/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved.

The laboratory undergoes internal standard testing. It also participates in GEDNAP blind trials for the aforementioned Y loci and is awarded with the respective proficiencies.

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


Table 1 Haplotype 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.

Fig. 1. Distribution of samples across the Greek Peripheries. The names of the Peripheries are described in Table 1.

The population fulfills the recommendations of the DNA Commission of the International Society of Forensic Genetics [16] and has been accepted in the Y chromosome Haplotype Reference Database with Accession Number YA003647. 6. Analysis of data Haplotype (gene) diversity values h were calculated using the formula [17]

X  N  1 p2i N1


where N is the population size (namely 200 subjects) and pi is the frequency of the ith haplotype. Standard error SE was also calculated according to the equation vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u8 nP P 2 2 o9 u< = pi p3i  u SE ¼ t 2 : ; N


In this work we have calculated the gene diversity for the whole population, as well as for each locus individually. The statistical analysis has been performed with Microsoft Office XP Excel. 7. Results and discussion A total of 200 subjects from Greece were investigated in this study. Analysis revealed 182 distinct haplotypes, with haplotype diversity 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 than once in this population. We can note also that only one haplotype was found five times, while all others appear three times or less. In our data the diversity of each locus is usually between four and seven alleles. Four allele variations appear in loci DYS389-I and DYS437, both of them having a single representation of allele 15 in the former, and allele 17 in the latter. Five alleles appear in loci DYS391, DYS392 and DYS438. Six alleles appear in loci DYS19, DYS389-II, DYS390 and DYS393, while seven alleles in locus DYS439. Locus DYS385 was found with 13 alleles, as we take into





Western Greece Attica Peloponnese East Macedonia and Thrace North Aegean Western Macedonia Hepirus Ionian Islands Central Macedonia Crete and Southern Aegean Sterea Ellada Thessaly


31 25 19 21 11 10 18 7 25 6 17 10

31 25 18 21 11 10 18 7 23 6 17 10

account that the gene DYS385 is duplicated and consists of two copies (DYS385a and DYS385b) [18–20]. For every locus the gene diversity has also been calculated and presented. The locus with the highest diversity is locus DYS385 with values h = 0.9537 and SE  0.00396. This is expected since this locus has the highest number of allelic variation and also corresponds to a duplicated gene. Locus DYS19 has values of h = 0.7671 and SE  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 gene diversity with value h < 0.7. The locus with the smallest gene diversity is DYS392 with values h = 0.3709 and SE  0.02689. The haplotypes found per Periphery is presented in Table 1. The observed discrepancy between the 182 distinct haplotypes in the whole data-set and the sum of distinct haplotypes per Periphery (197) is due to the fact that subjects from different Peripheries share the same haplotype. In most of the cases, no haplotype duplicity exists in our specimens, or if it exists, the duplicate haplotypes are very few. For this reason, the calculation of haplotype gene diversity is of no practical value. Genetic distance between neighbor populations has been estimated with the Analysis of Molecular Variance tool (AMOVA [23], YHDR database [24]). In Table 2 we present the calculated pairwise FST and p-values distances between the population presented here and other Greek populations; a population from Northern Greece [5] and a population from Continental Greece and the islands of Crete and Chios [7] have been taken into account. The parameters chosen are threshold of T FST ¼ 0:1, minimal size of cluster Smin = 10 and 10.000 permutations. Similar statistics have been performed between this Greek population and an Albanian population set of 8 population samples with 559 haplotypes, an Egyptian population set of 6 population samples with 291 haplotypes, an Italian population set of 40 population samples with 3555 haplotypes, a Spanish population set of 20 population samples with 2692 haplotypes and a Turkish population set of 13

Table 2 Calculated 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


N. Greece


Greece N. Greece CG/C/C

– 0.0003 0.0004

0.1616 – 0.0002

0.8631 0.1386 –

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


Table 3 Calculated 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

– 0.0048 0.0046 0.0064 0.0053 0.0036

0.0000 – 0.0005 0.0021 0.0009 0.0007

0.0000 0.0014 – 0.0010 0.0011 0.0008

0.0000 0.0000 0.0000 – 0.0027 0.0024

0.0000 0.0000 0.0000 0.0000 – 0.0013

0.0000 0.0005 0.0001 0.0000 0.0000 –

Fig. 2. Nonlinear Mapping For Data Structure Analysis for the Greek, Albanian, Egyptian, Italian, Spanish and Turkish population.

population samples with 1302 haplotypes, presented in Table 3. In Fig. 2 the Mapping for Data Structure Analysis (MDS plot) using Sammon mapping [21,22] is presented. The genetic distance between the Greek and the Italian and Egyptian population is smaller compared with the Turkish and Spanish populations, with the Albanian population to have the largest distance between these populations. The gene diversity of the Y chromosome related STRs has been found to be statistically important, in the presented population. This indicates that the usage of Y specific STRs are useful as a forensic evidence for the Greek population.

8. Other remarks This paper follows the guidelines for publication of population data requested by the journal [25].

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