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Genetic parameters and allele frequencies of five new European Standard Set STR loci (D10S1248, D22S1045, D2S441, D1S1656, D12S391) in the population of Romania

Aim To establish allele frequencies and genetic parameters for 5 new European Standard Set short tandem repeat (STR) loci in the population of Romania and to compare them with those in other populations. Methods DNA was isolated using QIAamp 96 DNA Swab BioRobot Kit and Chelex 100 methods. Polymerase chain reaction amplification was done using Investigator ESSplexPlus Kit (D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D10S1248, D12S391, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, TH01, and vWA). For DNA typing, Applied Biosystems 3500/3500xL Genetic Analyzer was used. Statistical analysis was done using Powerstats, GDA, and Arlequin software. Results Power of discrimination and polymorphism information content was highest for two new ESS loci, D1S1656 and D12S391. Comparison of allele frequencies for 5 new ESS loci in Romanian population with previously published population data showed significant differences for all compared populations, with the exception of Hungary. Geographically more distant populations, such as Spain, Sweden, United Kingdom, Germany, and Portugal differed more than closer populations. Conclusion New ESS STR loci are very useful for the analysis of forensic samples (persons or traces) due to their characteristics (shortness and high polymorphism). In comparisons with other common STR markers, they have a higher power of discrimination and also higher polymorphism information content, and could be used in any national DNA database.

The establishment of standard sets (or common sets) of short tandem repeat (STR) markers, which had first been a necessity for the forensic scientific community, as a result of globalization became a necessity for the worldwide law enforcement agencies. STR markers standard sets facilitate communication and judicial transmission of the forensic DNA typing results between different forensic groups or countries (1).
Although several STR sets have been proposed (2) Romania adopted the new 5 ESS loci and started to use them at the national level at the beginning of 2012 as part of the Investigator ESSplex Plus Kit, which replaced the AmpFlSTR Identifiler PCR Amplification Kit used before for Romanian National DNA Database supplying. As a consequence, and due to the lack of any study involving D10S1248, D22S1045, D2S441, D1S1656, and D12S391 loci in the Romanian population, allele distribution and genetic parameters of these loci have to be determined. The aim of this study was to establish allele frequencies and genetic parameters for 5 new ESS loci in population of Romania and to compare them with those in other populations .
The samples were taken from the Romanian National DNA Database. This database contains DNA samples collected from convicted offenders, required to provide DNA samples in accordance with the 76/2008 law.

DnA analysis
DNA from 1331 buccal swabs (saliva) was extracted using QIAamp 96 DNA Swab BioRobot KIT on a BioRobot Universal System (Qiagen, Hilden, Germany), according to the manufacturer's instructions, and occasionally (when samples had to be manually processed) using Chelex 100 method (5). The human DNA obtained from collected samples was quantified using Investigator Quantiplex Kit (Qiagen) and a 7500 Fast Real-Time PCR Systems (Applied Biosystems, Foster City, CA, USA). Approximately 1 ng target DNA from each sample was amplified using the Investigator ESSplex Plus Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions and 9600 Thermal Cyclers (Applied Biosystems). Electrophoresis, and genotyping of amplicons were carried out on the Applied Biosystems 3500/3500xL Genetic Analyzer using GeneMapper ID-X software.

Statistical analysis
For all autosomal 15 loci of Investigator ESSplex Plus Kit, observed heterozygosity, expected heterozygosity, and Pvalues of the Hardy-Weinberg equilibrium tests were assessed using GDA version 1.1 (6) and also matching probability, power of discrimination, polymorphism information content, probability of exclusion, and typical paternity index were calculated with a modified version of Powerstats Version 1.2 (7). Population differentiation tests were carried out using allele frequencies from previously published population data for the new five ESS loci (Austria, Belgium, Croatia, Czech, Germany, Hungary, Italy, Macedonia, Poland, Portugal, Spain, Sweden, and United Kingdom) using Arlequin Software version 3.1.1 (8).
The allele frequency distributions for the 15 STR markers studied in Romanian population and statistical parameters (Hardy-Weinberg equilibrium -P, expected heterozygosity, observed heterozygosity, matching probability, power of discrimination, polymorphism information content, probability of exclusion, and typical paternity index) are summarized in  (17); at two loci with the population from the Czech Republic (12) and Macedonia (16); at three loci with the population from Belgium (10); at four loci with the population from Spain (19), Sweden (20), and United Kingdom (21); at five loci with the population from southern part of Germany (13) and Portugal (18).

DISCuSSIon
For the five new 5 ESS loci, there were no significant differences between the population from Romania and Hungary (14), there was a moderate number of differences between Romania and Austria, Croatia, Italy, Poland, Macedonia, and the Czech Republic, and there was a great number of differences with the rest of the countries used for allele frequencies comparison.
Based on the number of differentiations (number of loci), it can be concluded that populations differ according to the geographic location. So, geographically distant populations, such as Spain (19), Sweden (20), United Kingdom (21), Germany (13), and Portugal (18), differed  more from the Romanian population than relatively closer populations.
It seems that due to higher power of discrimination and higher polymorphism information content, these ESS markers are very good in discriminating between geographically different populations. This implies that they are very useful in human identification and therefore each country that has a national DNA database should establish their allele frequencies in order to use them for forensic purposes at the national or international level.
The analysis of the new 5 ESS STR loci extended the total number of different STR markers analyzed for Romanian population and to a certain degree confirmed the results of population comparisons done using other STR markers in four other Romanian smaller geographical and historical regions: Moldova (22), Transylvania (23), Wallachia (24), and Dobruja (25).
In conclusion, new ESS STR loci are very useful for the analysis of forensic samples (persons or traces) due to their characteristics (shortness and high polymorphism). In comparisons with other common STR markers they have a higher power of discrimination and also higher polymorphism information content. They are very helpful in daily routine of National DNA Database and should be used as a common procedure.
Funding This research was supported by the General Inspectorate of Romanian Police.
ethical approval Granted by the institution.
Declaration of authorship FS was in charge of technical organizational aspects, performed a part of the experimental work, interpreted the data, conducted statistical analysis, and worked on the manuscript from the early beginning until the final version. SV, VC, and FI performed a part of the experimental work, interpreted the data, and participated in manuscript design, preparation, and verification. DC, AC, and IMS performed a part of the experimental work and interpreted the data.