A panel of tetranucleotide STR markers as an alternative approach to forensic DNA identification of wolf and dog.

Commercial panels of microsatellite (STR) loci are focused on the use of DNA of the domestic dog (Canis lupus familiaris) and are often inapplicable for genotyping the DNA of the gray wolf (Canis lupus lupus). We propose a CPlex test system, including one hexa- and 12 tetranucleotide autosomal STR loci, as well as two sex loci, that is equally efficient in DNA identification of biological samples of the wolf and the dog. Analysis of molecular variance between samples revealed significant differentiation values (FST = 0.0784, p < 0.001), which allows to use the panel to differentiate wolf and dog samples. Population subdivision coefficients (θ-values) were calculated for each of the 13 STR loci of the developed test system. It was shown that the values of the genotype frequency for dogs and wolves, without and with considering the θ-value, differ by three orders of magnitude (for dogs 8.9 × 10-16 and 2.1 × 10-14 and for wolves 1.9 × 10-15 and 4.5 × 10-14, respectively). The use of population subdivision coefficients will allow to identify the most reliable results of an expert identification study and the power of exclusion provided by the STR loci of the CPlex test system makes it possible to achieve a reliable level of evidence in forensic DNA analysis of both wolves and dogs. The test system has been validated for use in forensic identification of the dog and wolf based on biological traces found at crime scenes, as well as for individual identification and establishing biological relationship of animals of these species.

STR Profiling for Discrimination between Wild and Domestic Swine Specimens and between Main Breeds of Domestic Pigs Reared in Belarus.

A panel comprising 16 short tandem repeats (STRs) and a gender-specific amelogenin marker was worked out and tested for robustness in discrimination between wild and domestic swine subspecies encountered in Europe, between regional populations of wild boars and between main breeds of domestic pigs reared in Belarus. The STR dataset comprised 310 wild boars, inhabiting all administrative regions of Belarus, and 313 domestic pigs, representing three local and three cosmopolitan lines. Additionally, a total of 835 wild boars were genotyped for the presence of melanocortin 1 receptor (MC1R) alleles specific for domestic pigs. Correctness of assignment of STR profiles to appropriate populations was measured by log-likelihood ratios (log-LRs). All samples were correctly identified as wild boars or domestic pigs with average log-LR of 42.4 (LR = 2.6×1018). On the other hand, as many as 50 out of 835 (6.0%) genotyped wild boars from Belarus possessed MC1R alleles specific to domestic pigs, demonstrating supremacy of our STR profiling system over traditional differentiation between wild boars and domestic pigs, based on single binary markers. Mean log-LRs for allocation of wild boars to their regions of origin and of domestic pigs to appropriate breeds were 2.3 (LR = 9.7) and 13.4 (LR = 6.6×105), respectively. Our results demonstrate the developed STR profiling system to be a highly efficient tool for differentiation between wild and domestic swine subspecies and between diverse breeds of domestic pigs as well as for verification of genetic identity of porcine specimens for the purpose of forensic investigations of wildlife crimes, assurance of veterinary public health, parentage control in animal husbandry, food safety management and traceability of livestock products.

Variation of X-chromosomal microsatellites in Belarus within the context of their genetic diversity in Europe.

More and more X-STR data are becoming available for worldwide human populations for forensic and anthropological investigations, but the European datasets analysed so far represent mainly the central, northern, western and southern part of the continent with populations of Eastern Europe being practically uninvestigated. In the present study, we assessed genetic variation and linkage disequilibrium of 19 X-chromosomal STR markers (DXS7132, DXS7133, DXS7423, DXS7424, DXS8377, DXS8378, DXS9895, DXS10074, DXS10075, DXS10079, DXS10101, DXS10103, DXS10134, DXS10135, DXS10146, DXS10147, DXS10148, GATA172D05, HPRTB) in four regional populations of an Eastern European state of Belarus, including 12 loci incorporated in the Argus X-12 kit. Our results revealed cumulative power of discrimination of the tested X-STR loci to amount to 0.999999999999996 and 0.999999997 in females and males, respectively. Analysis of molecular variance demonstrated regional stratification within the country, excluding the use of a common X-STR database for Belarus in forensic casework. However, development of a separate X-STR database for the northwestern part of the country or exclusion of four loci displaying regional differences from the dataset were shown to eliminate the observed geographic substructure among Belarusians. Comparison of the Belarusian genotypes with X-STR data from other European populations disclosed a geography-driven northeast-southwest gradient extending from Belarus and Finland to Iberia and Italy. This study is the first extensive report on variation of X-STR markers in populations from Eastern Europe and the first comprehensive analysis of diversity of X-chromosomal microsatellites in Europe.

[Identification of a novel Y-SNP in the USP9Y gene and its impact on genotyping alleles of the M46 locus]. / Identyfikacja polimorfizmu typu Y-SNP w genie USP9Y i jego znaczenie w genotypowaniu alleli locus M46.

Y-chromosomal SNP markers are becoming increasingly more popular among forensic geneticists, but since they constitute variants specific to the ethnic origin, detailed population studies are required. Research into frequency of haplogroup N-M46 in the Belarusian population detected a mutated allele in 22 males, including one with a very distinct Y-STR haplotype. Sequencing of the M46 locus of this individual revealed the presence of a novel Y-SNP nearby the M46 locus, which was responsible for the erroneous assignment of the Y chromosome to the haplogroup N-M46. An impact of the identified polymorphism on discrimination of alleles of the M46 locus with various techniques was discussed, and solutions ensuring correctness of the genotyping results were proposed.

Forensic analysis of polymorphism and regional stratification of Y-chromosomal microsatellites in Belarus.

Nine loci defining minimal haplotypes and four other Y-chromosomal short tandem repeats (Y-STRs) DYS437, DYS438, DYS439 and GATA H4.1 were analysed in 414 unrelated males residing in four regions of Belarus. Haplotypes of 328 males were further extended by 7 additional Y-STRs: DYS388, DYS426, DYS448, DYS456, DYS458, DYS460 and DYS635. The 13-locus haplotype diversity was 0.9978 and discrimination capacity was 78.7%, indicating presence of identical haplotypes among unrelated males. Seven additional Y-STRs enabled almost complete discrimination of undifferentiated 13-locus haplotypes, increasing haplotype diversity to 0.9998 and discrimination capacity to 97.9%. Analysis of molecular variance of minimal haplotypes excluded the use of a Y-STR database for Belarusians residing in northeastern Poland as representative for the Belarusian population in forensic practice, and revealed regional stratification within the country. However, four additional markers (DYS437, DYS438, DYS439 and GATA H4.1) were shown to eliminate the observed geographical substructure among Belarusian males. The results imply that in case of minimal and PowerPlex Y haplotypes, a separate frequency database should be used for northern Belarus to estimate Y-STR profile frequencies in forensic casework. In case of Yfiler haplotypes, regional stratification within Belarus may be neglected.

A reference data base on STR allele frequencies in the Belarus population developed from paternity cases.

The study considers data on 15 STR-loci from paternity cases across Belarus districts from 2004 to the beginning of 2006 (set #1, 580 individuals) and since that to the beginning of 2007 (set #2, 530 individuals); the data majorly consist of ethnic Belarusians. No significant difference was found between the sets, as well as between the country districts in which the cases occurred. The allele frequencies obtained are very similar to those based on population survey at common loci. Therefore, a data base can be constructed of data from wide survey on paternity cases. Pooling the sets together provides a reference data base on the Belarus population. Additionally, we compared the allele profiles to those in other Slavic groups from the former USSR: Russians from Moscow and eastern Ukrainians from Kharkov city.

Analysis of forensically used autosomal short tandem repeat markers in Polish and neighboring populations.

The purpose of this study was to evaluate the homogeneity of Polish populations with respect to STRs chosen as core markers of the Polish Forensic National DNA Intelligence Database, and to provide reference allele frequencies and to explore the genetic interrelationship between Poland and neighboring countries. The allele frequency distribution of 10 STRs included in the SGMplus kit was analyzed among 2176 unrelated individuals from 6 regional Polish populations and among 4321 individuals from Germany (three samples), Austria, The Netherlands, Sweden, Czech Republic, Slovakia, Belarus, Ukraine and the Russian Federation (six samples). The statistical approach consisted of AMOVA, calculation of pairwise Rst values and analysis by multidimensional scaling. We found homogeneity of present day Poland and consistent differences between Polish and German populations which contrasted with relative similarities between Russian and German populations. These discrepancies between genetic and geographic distances were confirmed by analysis of an independent data set on Y chromosome STRs. Migrations of Goths, Viking influences, German settlements in the region of Volga river and/or forced population resettlements and other events related to World War II are the historic events which might have caused these finding.

Y-STR variation among Slavs: evidence for the Slavic homeland in the middle Dnieper basin.

A set of 18 Y-chromosomal microsatellite loci was analysed in 568 males from Poland, Slovakia and three regions of Belarus. The results were compared to data available for 2,937 Y chromosome samples from 20 other Slavic populations. Lack of relationship between linguistic, geographic and historical relations between Slavic populations and Y-short tandem repeat (STR) haplotype distribution was observed. Two genetically distant groups of Slavic populations were revealed: one encompassing all Western-Slavic, Eastern-Slavic, and two Southern-Slavic populations, and one encompassing all remaining Southern Slavs. An analysis of molecular variance (AMOVA) based on Y-chromosomal STRs showed that the variation observed between the two population groups was 4.3%, and was higher than the level of genetic variance among populations within the groups (1.2%). Homogeneity of northern Slavic paternal lineages in Europe was shown to stretch from the Alps to the upper Volga and involve ethnicities speaking completely different branches of Slavic languages. The central position of the population of Ukraine in the network of insignificant AMOVA comparisons, and the lack of traces of significant contribution of ancient tribes inhabiting present-day Poland to the gene pool of Eastern and Southern Slavs, support hypothesis placing the earliest known homeland of Slavs in the middle Dnieper basin.

Belarusian population genetic database for 15 autosomal STR loci.

Allele frequencies of 15 short tandem repeat loci included in the AmpFlSTR Identifiler kit (Applied Biosystems) were obtained from a sample set of unrelated individuals living in Belarus (n=176). For all loci, no deviation from Hardy-Weinberg equilibrium was found. Results were compared with data available for the Belarusian minority residing in northeastern Poland and for other Slavic populations. Statistically significant differences were observed between Belarusians and all compared populations. The values of heterozygosity, polymorphic information content (PIC), power of discrimination (PD), power of exclusion (PE), paternity index (PI) and matching probability (pM) were calculated.

A comprehensive population survey on the distribution of STR frequencies in Belarus.

This work develops a detailed STR database from 11 population samples and samples from paternity analyses from different districts of Belarus. The combined data on 2020 individuals form a total database for the country, with the exclusion power of 99.987% based on 11 STR loci. Possible differentiation in allele frequencies between population samples, small in terms of F-statistics and undetectable by standard statistical tests, is discussed.