An investigation of 10 Y-STR loci and the detection of specific haplotype frequencies in Turkish population

This study is to survey 10 Y-STR loci in 241 males from Turkey. In this study, the 241 healthy and unrelated males living in different parts of Turkey for at least three generations were included. Genomic DNAs were isolated from peripheral blood samples by standard phenol-chloroform extraction method. 10 Y-STR loci including DYS19, DYS385a/b, DYS388, DYS389I/II, DYS390, DYS391, DYS392, DYS393, and YCAIIa/b were analyzed by using PCR and denaturing PAGE. Allele frequencies, gene diversities and haplotype frequencies were analyzed. Gene diversity per locus varied from 0.5788 (DYS388) to 0.8903 (DYS385a/b). The numbers of haplotypes in minHt recommended by YCC and Ht10 have been 208 and 186, respectively. When our minHt haplotypes frequencies compared with the other seven populations, we have found statistically significant differences between our results and other populations (p < 0.01) except that Czech population (p > 0.05). We suggest that an alternative haplotype designated as aHt maybe alternative to minHt in respect of its Y-STR content with the highest gene diversity value. The aHt haplotype has found a higher discriminatory potential than minHt haplotype with a better Pd combined value (0.9999936 vs 0.9999836) and has higher average gene diversity per locus (0.7834 vs 0.7518) in Turkish population. aHt haplotype can be proposed as an alternative to minHt in paternity testing and forensic medicine applications involving Turkish male population. This study has also provided additional information to the framework of variation involving 10 Y-STR loci as well as a further contribution to the Y-STR database for Turkish male population.


INTRODUCTION
Human Y chromosome has been known to display comparatively low levels of polymorphisms in contrast with autosomal chromosomes.Nonetheless, there are many types of Y-chromosome specific polymorphisms identified in the non-recombining region of Y (NRY) including RFLPs, Y Alu and microsatellite polymorphisms [1,2].Microsatellites or short tandem repeats (STRs) are dispersed throughout the genome and are known to be highly polymorphic; hence, each microsatellite locus generally has many alleles due to variable number of repeat units [3,4].
Allelic genotyping of STRs does not require the use of complex molecular techniques, since amplifications and visualization of PCR products make it easy.Y-chromosome specific STRs (Y-STRs) are chosen as more informative in paternity testing, forensic applications and the study of population histories due to the haploid state of Y chromosome which ensures both the transmittance by the paternal lineages and the lack of recombination in NRY, excluding pseudoautosomal regions (PARs) [5][6][7][8][9][10][11].
Allelic and haplotypic distributions of Y-STRs have shown significant differences in different geographical regions, ethnical groups and communities [12][13][14][15][16][17][18].Therefore, allelic and haplotypic frequencies of Y-STRs should be determined in a male population prior to any interpretations of forensic analysis and paternity testing [6,[8][9][10][11]19].In this study, allelic and haplotypic frequent-cies involving 10 Y-STR loci: 8 Y-STR loci as recommended by Y Chromosome Consortium (YCC) plus DYS388 and YCAIIa/b-have been determined with such a necessity in a representative group of Turkish population in order to make comparisons with other populations.
Healthy and unrelated 241 males living in different parts of Turkey for at least three generations were included in this study.The written informed consents were obtained from the study subjects and the study protocol was approved by the ethics committee of Ankara University Medical Faculty.
Genomic DNAs were isolated from peripheral blood samples by standard phenol-chloroform extraction method [22].Four multiplex PCR analyses were carried out with locus specific primers in a total volume of 25 µl reaction mixture, containing; 50 -150 ng genomic DNA, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl 2 , 0.1 -0.8 µM of each primer, 200 µM of each dNTP (Sigma), 5 µg BSA and 1 unit Taq DNA polymerase (Invitrogen) [23].Y-STRs amplified in combination in multiplex PCR are as follows: DYS389I, DYS389 II and DYS390 in multiplex I; DYS392 and DYS393 in multiplex II; DYS19 and DYS388 in multiplex III; DYS385a/b and DYS391 in multiplex IV; while YCAIIa/b loci were amplified separately.Cycling conditions were as follows: 32 cycles of 94˚C -1 min, 54˚C -1 min, 72˚C -1 min for multiplex I, II, IV and YCAIIa/b; 35 cycles of 94˚C -1 min, 55˚C -1 min, 72˚C -60 min for multiplex III.An initial denaturation at 94˚C -2.5 min and a final extension at 72˚C -10 min were performed before and after each cycling reactions.Female DNA was used as a negative control in every run.
Amplified products were separated by 6% denaturing polyacrylamide gel electrophoresis (PAGE) for 3 h at 1500 V. Visualization of PCR products was carried out by a modification of the silver staining method of Santos, et al. [23].Gels were fixed for 15 min at room temperature in 10% (v/v) ethanol which were treated with 1% (v/v) nitric acid for 3 min with agitation thereafter.Gels were then rinsed in deionized water for 1 min and treated with 0.2% (w/v) silver nitrate and 0.1% (v/v) formaldehyde solution for 25 min with agitation.Gels were rinsed for a few seconds in deionized water, and developed in an aqueous solution of 3% Na 2 CO 3 (w/v), 100 µl/L 2% (w/v) Na 2 S 2 O 3 and 0.1% formaldehyde until the bands were well visualized.Staining was ended with a fixative solution.
Allele frequencies, gene diversities (H), haplotype frequencies and genetic differentiation between populations were computed using Arlequin 3.1.1software.Allele frequencies were calculated by gene counting, while H was computed for each locus according to the by formula (1), where n is the number of samples, k is the number of haplotypes, and p i is the frequency of i-th haplotype [24].
The Combined Power of Discrimination (P d combined ) for haplotypes was calculated using the by formula (2), where P di is Power of Discrimination of i-th locus [25].

RESULTS AND DISCUSSION
10 Y-STRs have been analyzed for diversity in 241 healthy and unrelated male individuals from Turkey.Observed allele or genotype frequencies of the 10 Y-STR loci have been given in Table 1.Variations in the number of individuals for certain loci have been brought about by some technical problems not anticipated.Gene diversity values for each 10 Y-STR loci have been given in Table 1.The lowest gene diversity (0.5788) has been found in DYS388 locus, wherein the most frequent allele has been allele 13 with a frequency of 62.08%.This result has been in accord with the data reported by YCC [21].The highest gene diversity (0.8903) has been found in DYS385 locus, wherein the most frequent allele has been allele 14 with a frequency of 17.62% (Table 1).
The observed number of haplotypes and their frequencies involving minHt and Ht10 haplotypes in this current survey have been tabulated in Table 2.The number of haplotypes detected for minHt is 208 and 186 for Ht10.Each haplotype belonging to Ht10 have been found to be unique while the same holds for minHt with the exception of H15 which has been detected in two individuals.Gene diversity, average gene diversity per locus and Combined Power of Discrimination (P d combined ) values for Ht10 and minHt haplotypes have been given in Table 3.
Our results suggest that an alternative haplotype (aHt), which differs slightly from minHt in respect of its Y-STR loci contents, maybe alternative for minHt in Turkish population.aHt has included the selected 8 Y-STR loci: DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS392, DYS393, and YCAIIa/b.The only difference between the minHt and the proposed aHt is the inclusion of YCAIIa/b locus in place of DYS391 locus in aHt due to its higher gene diversity value of 0.8087 as compared with 0.6101 of DYS391 locus (Table 1).We have found 186 unique haplotype in aHt (data not shown).The aHt has reflected a better P d combined value when compared with minHt (0.9999936 vs 0.9999869) and, has higher average gene diversity per locus (0.7834 vs 0.7518) (Table 3).The data has exhibited that aHt has a higher discriminatory potential than that of minHt.

Table 1 .
Detected allele frequencies and gene diversities of the Y-STR loci in Turkish population.
* Allele frequencies was calculated including two genomic copies.

Table 2 .
Detected number of haplotypes and their frequencies in minHt and Ht10 haplotypes surveyed in Turkish population in this study.

Table 3 .
Comparative presentation of haplotype numbers, gene diversities, average gene diversities and P d combined values belonging to three haplotypes surveyed in this study.
*Combined Power of Discrimination.