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Institute of Medicine and Law Department of Anatomical, Histological, Forensic and Orthopaedic Sciences, Sapienza University of Rome, IRCSS Neuromed, Pozzilli, Italy
The 17 Y-STR loci included in the AmpFLSTR Yfiler PCR Amplification Kit were analyzed in 98 unrelated healthy males from Apulia (Southern Italy). A total of 97 different haplotypes were identified, of which 96 haplotypes were unique and 1 occurred twice. Allele frequencies for each Y-STR locus in pooled sample and estimated value of gene diversity (GD) were evaluated. The lowest value of GD was observed for DYS392 (0.126) and the highest one (0.936) for DYS385. The HD (haplotype diversity) for the studied Y-STR set showed a value of 0.9994, with an HMP (haplotype match probability) value of 0.0006, while the overall DC was 98.98%. Microvariant alleles were found for the DYS458 and DYS385 markers and sequenced.
Furthermore, Φst-based genetic distance computation and pair-wise analysis of molecular variance (AMOVA) test were carried out. When comparing our population with the Apulia sample previously investigated, the AMOVA analysis detected no evidence for significant differentiation. The comparison with all Italian populations submitted to the YHRD website showed no relevant differences with all Southern Italian populations (San Giorgio La Molara, Belvedere, Trapani and Catania) and significant genetic deviation with all Northern Italian populations (Udine, Biella, La Spezia, Modena, Ravenna, Marche and North Sardinia). Moreover, the other populations and meta-populations belonging to the whole Mediterranean area (Croatia, Macedonia, Albania, Greece, Turkey, Israel, Libya, Tunisia, Algeria, Morocco and Spain) were different from our Apulia sample. The data were submitted to YHRD.
We evaluated allele and haplotype frequencies as well as statistical forensic parameters in a sample of 98 unrelated healthy males from Apulia region (Southern Italy) for 17 Y-STRs, using AmpFLSTR Yfiler PCR Amplification Kit (Applied Biosystems). This multiplex PCR reaction permits to amplify the loci of the European minimal haplotype (DYS19, DYS385 a/b, DYS389 I/II, DYS390, DYS391, DYS392, DYS393), the loci recommended by SWGDAM (including European minimal haplotype plus DYS438 and DYS439) and additional highly polymorphic loci (DYS437, DYS448, DYS456, DYS458, DYS635 and Y GATA H4).
Blood samples and oral swab were collected after informed consent from all individuals. Genomic DNA was extracted from peripheral blood and buccal cells transferred on FTA Cards (Whatman) by Whatman FTA purification reagents. After purification, a 1.2-mm bloodstained and buccal-swab-stained punch, containing approximately 5–20 ng DNA, was directly added to the PCR mix and amplified. The PCR amplification was carried out in a GeneAmp PCR 9700 Thermal Cycler (Applied Biosystems) according to the manufacturer's instructions for the AmpFLSTR Yfiler PCR Amplification Kit. Separation and detection of PCR products were performed on the ABI Prism 310 Genetic Analyzer (Applied Biosystems). Genotyping of each sample was carried out automatically using the allelic ladder provided with the AmpFLSTR Yfiler PCR Amplification Kit and using GeneMapper® ID software v3.2 (Applied Biosystems). After the initial genotyping, DNA samples with microvariant alleles were sequenced using Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The DNA control 007 included in the AmpFLSTR Yfiler PCR Amplification Kit was used as quality control template. The list of primer sequences employed for the sequencing reactions [
Allele and haplotype frequencies were computed using the gene counting method. Gene diversity (GD) and haplotype diversity (HD) were calculated according to Nei [
]. Haplotype match probability (HMP), that is the probability of finding an identical haplotype in a pair of randomly unrelated males, was calculated as HMP = 1 − HD. The discriminatory capacity (DC) was determined dividing the total number of different haplotypes by the total number of individuals in the population analyzed. Pair-wise genetic distances between populations were carried out based on Φst and the significance was tested with 10,000 permutations using AMOVA tool provided by YHRD website (www.yhrd.org) [
] population samples. However, previously published studies regarding Apulia population were only based on 8 Y-STRs, while the present study investigated on a panel of 17 Y-STRs. We identified a total of 97 different haplotypes in a sample set of 98 unrelated individuals, with 96 haplotypes being unique and 1 occurring twice (see Supplementary Table 2). Allele frequencies and estimated values of gene diversity (GD) for each Y-STR locus are presented in Table 3, available as e-component. The lowest value of GD was observed for DYS392 (0.126), while the highest one (0.936) is presented by DYS385. The HD for the studied Y-STR set, corresponding to the chance of exclusion for unrelated males, showed a value of 0.9994, with an HMP value of 0.0006, while the overall DC was 98.98%.
Furthermore, microvariant alleles were found for the DYS458 marker, such as 17.2 (1 individual) and 18.2 (3 individuals) alleles, and in the DYS385 marker, namely the 16.3 allele (1 individual). Sequencing of these samples confirmed the insertion of two and three nucleotide pairs in these alleles, respectively. Nomenclature for these alleles was according to the International Society of Forensic Genetics (ISFG) guidelines [
]. In our population we also observed the 19 allele of 242 bp at DYS635 locus in one sample. This allele is not included in the allelic ladder provided with the AmpFLSTR Yfiler PCR Amplification Kit, but it has been found in other population studies [
], the AMOVA analysis detected no significant differences (p value = 0.9213). Φst-Based genetic distance was also assessed among our Apulia population and populations from South and North-West Apulia previously described elsewhere [
]. Also in this case, no evidence for significant differentiation has been detected (Φst < 0.08).
Moreover, we compared Apulia Y-STR data with all populations and metapopulations belonging to the whole Mediterranean area, both Italian and non-Italian, thus-far submitted to the YHRD database. As far as concerns the comparisons with the Southern Italian populations, our analysis showed no genetic deviation (p = 0.0664 with San Giorgio La Molara, p = 0.6546 with Belvedere, p = 0.0768 with Trapani, p = 0.7383 with Catania) [
]. By contrast, AMOVA analysis indicated significantly differences (p < 0.05) with respect to all Northern Italian populations (Udine, Biella, La Spezia, Modena, Ravenna, Marche and North Sardinia) [
]. Regarding the non-Italian populations belonging to the Mediterranean basin, AMOVA analysis showed that our Apulia sample had significant differences with all the populations included in YHRD (Croatia, Macedonia, Albania, Greece, Turkey, Israel, Libya, Tunisia, Algeria, Morocco and Spain) [
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