| Objective:Hazara population across Durand-line has experienced extensive interaction with central Asian and eastern Asian populations.Hazara individuals have typical Mongolian facial features and they claim to be descendants of Genghis Khan‘s army in the first quarter of the thirteenth century AD.Previously,worldwide analysis of Y-chromosomal haplotype diversity for rapidly mutating(RM)Y-STRs and with PowerPlex?Y23 System(Promega Corporation Madison,USA)kit were created with collaborative efforts,but Hazara population from Pakistan and Afghanistan were missing.Previous study based on limited number of Y STRs with limited number of samples showed that Hazaras are descendants of Mongols.Dispute about who Hazaras are still continues and there are three theories.First theory is―they could be Mongol-Turk origin,descendants of an occupying army left in Afghanistan by Genghis Khan‖.Second theory goes back to area of Kushan Dynasty,when Bamiyan in Afghanistan was central stage for Buddhist civilization.Believers of this theory support their logic by saying that―Hazara people have similar facial structures with Buddhist murals and statues in the region‖.Third and most accepted theory is that―Hazara are the mixed race‖.Thus our aim is to check the potential forensic usefulness of the 27 Y-STRs studied and also shed light on the population history of Hazara population.Methods:A total of 413 blood samples were collected,in which 153 from Hazara Town Quetta,Baluchistan Pakistan(Participants were part of an earlier study and were agreed to the secondary use of their DNA samples)and 260 from Bamyan,Afghanistan.The ethical review committee of China Medical University,Shenyang,Liaoning Province,and P.R.China approved the project and obtaining of samples.Genomic DNAs were extracted from blood using QIAamp?DNA Blood kit(Qiagen,Germany)according to the manufacturer‘s instructions.Yfiler?Plus PCR Amplification Kit(Thermo Fisher Scientific)which contains(DYS19,DYS385a/b,DYS389I/II,DYS390,DYS391,DYS392,DYS393,DYS437,DYS438,DYS439,DYS448,DYS456,DYS458,DYS635,Y GATA H4,DYS460,DYS481,DYS533DYF387S1a/b,DYS449,DYS518,DYS570,DYS576 and DYS627).).PCR amplification was carried out using Applied Biosystems○R GeneAmp○R PCR System9700 thermal cyclers.PCR amplifications were performed as recommended by the manufacturer,although using half of the recommended reaction volume(12.5?l).PCR products were detected and separated by capillary electrophoresis with ABI 3500Genetic Analyzer(Applied Biosystems,USA)according to manufacturer‘s instructions.The raw data were analyzed using Gene Mapper v.3.2.(Applied Biosystems,USA).54 Hazara samples showed DYS448 null alleles(detected as allelic dropouts)in three Y STR kits(GoldenEye Y26,Microreader?Y29 and Yfiler?Plus).The region was amplified using a nested PCR to reduce unspecific PCR products.Allelic frequencies were calculated through simple count method.Nei‘s formula was used to calculate gene diversity(GD)which is equivalent to haplotype diversity(HD)for Y STRs.Y STR data generated using Goldeneye?26Y kit and the Yfiler?Plus kit allowed the development of haplotype diversities(HD)were calculated for the 26/30 Y STR loci included in these kits and also the haplotype diversities for the minimal(9 loci);extended(11 loci);Powerplex Y(12 loci);Yfiler?(17 loci)and Powerplex Y 23(23 loci).An extended haplotype comprising of Yfiler?Plus with three additional loci(30 loci)was also developed and haplotype diversity calculated.Discrimination capacity(DC)was calculated as the ratio of unique haplotypes in the sample.Match probabilities(MP)were calculated asΣPi~2,where Pi is the frequency of the i-th haplotype.Genetic distances between Hazara population and other world populations were calculated using molecular variance(AMOVA)and multidimensional scaling(MDS).Both these analyses were performed using YHRD online tools(http://www.yhrd.org)based on pairwise Rst values.A neighbor-joining phylogenetic tree was constructed for the Hazara and the reference populations based on a distance matrix of Rst using the Mega7 software.Using the program Network 4.1.1.2.(http://www.fluxus-engineering.com/sharenet.htm),median joining network was constructed from data of Hazara population for 23 Y STRs(DYS19,DYS389II-I,DYS390,DYS391,DYS392,DYS393,DYS437,DYS438,DYS439,DYS456,DYS458,DYS635,Y_GATA_H4,DYS576,DYS570,DYS481,DYS533,DYS549,DYS643,DYS460,DYS449 and DYS388).Linear discriminant analysis(LDA)was performed on Hazara,Central Asia,East Asia;Russian and South West Asian samples using R program and correlation between STRs were calculated using XLSTAT(http://www.xlstat.com/en/).Results:Y-STR haplotypes from 413 male individuals in Hazara population were developed.The data showed 54 null alleles for DYS448.380(out of 413)different haplotypes were observed for 27 Y STRs.Allelic frequencies and GD values of various sets of Y-STR loci in Hazara individuals were calculated.240 alleles were observed for 27 Y STRs.DYF387S1 showed highest heterozygosity in both Hazara populations from Afghanistan and Pakistan with 0.9242 and 0.8792,respectively.Among single Y-STR markers DYS449,DYS627,DYS391,and DYS437 showed the highest or lowest heterozygosities with 0.8468,0.7949,0.3916 and 0.2383,respectively in both Hazara populations from Afghanistan and Pakistan.After pooling Hazara populations together DYF387S1,DYS437 showed the highest or lowest heterozygosities with 0.9257 and 0.4053 respectively.In Hazara population from Afghanistan we have observed 240 different alleles on 27 STRs while in Hazara population from Pakistan in 153 individuals we have observed 188 alleles on 27 Y STRs and 204 alleles on 30 Y STRs.Allelic frequencies ranged from 0.0038 to 0.6654in Hazara population of Afghanistan and 0.0065 to 0.8627 in Pakistani Hazara population.We evaluated forensic parameters at seven levels,the minimal 9 Y-STRs loci,the extended 11 Y-STRs loci,PowerPlex Y12 STRs loci,Y-filer 17 STRs loci,Y21STRs loci,Y27 Yfiler Plus loci,and 6 rapidly mutating Y STRs loci.The discrimination capacity(DC)ranged from 47.06%(the minimal 9 Y-STRs loci)to99.35%(Y27 Yfiler Plus loci)and haplotype diversity(HD)ranged 0.9316(the minimal 9 Y-STRs loci)to 0.9999(Y27 Yfiler Plus loci)in Pakistani Hazara population while DC ranged 41.15%(the minimal 9 Y-STRs loci)to 88.46%(Y27Yfiler Plus loci)and HD ranged 0.9708(the minimal 9 Y-STRs loci)to 0.9937(Y27Yfiler Plus loci)for Hazara population from Afghanistan.Pooling both populations together DC ranged 40.19%(the minimal 9 Y-STRs loci)to 92%(Y27 Yfiler Plus loci)and HD ranged 0.9689(the minimal 9 Y-STRs loci)to 0.9992(Y27 Yfiler Plus loci).Interestingly six rapidly mutating Y STRs which are included in Yfiler plus kit,detects high haplotype diversity.We have observed 139(90.84%)different haplotypes out of 153,among them 131(85.62%)were unique in Pakistani Hazara population while in Afghani Hazara population observed haplotypes were 188(72.30%)out of260,among them 152(58.46%)were unique.These six STRs(RM Y STRs)showed almost same diversity,shown by PPY 23 loci.To assess the relationship between the Hazaras of Pakistan and Afghanistan and the other populations residing within countries and neighboring regions,we have compared our populations data set with other population sets available in the YHRD by calculating pair-wise genetic distances.The AMOVA results suggested that the calculated Rst values between Hazara and South Asian populations.Afghan population(Rst=0.0381),Afghan Hazara population(Rst=0.0473)and Turk people from Ardabil,Iran(Rst=0.0868)showed the closest genetic distance while Zoroastrian population from Yazd,Iran(Rst=0.4451),Riang population from Tripura,India(Rst=0.4988)and Munda population from Jharkhand,India(Rst=0.5324)showed the greatest genetic distance from the Hazara population.In East Asia,Central Asia and Russian Federation Hazara population showed closest genetic distance between Kazakh population from Kazakhstan(Rst=0.0399),Uyghur population from Turpan,China(Rst=0.0523)and Yellow Uyghur population from Gansu,China(Rst=0.0603)while Miao population from Hunan,China(Rst=0.3579),Dai population from Xishuangbanna,China(Rst=0.3728)and Yao population from Liannan,China(Rst=0.4597)showed the greatest genetic distance from the Hazara population.We also have constructed neighbor joining(NJ)trees based on Fst values between Hazara population and 93 previously published populations from South Asia to assess the evolutionary relationships.Hazara population from Pakistan and Afghanistan are clustered with Afghan population from Afghanistan,Pashtun population from KPK,Pakistan.After this we have compared Hazara population with previously published49 populations from East Asia(China,Japan,South Korea and Mongolia),Central Asia and Russian Federation.Hazara populations are clustered with Kazakhs,Uyghurs,Kyrgyz and Mongols.The Y haplogroups were predicted using the online Y-haplogroup predictor software(http://www.nevgen.org/).C2(previously known as C3-Star cluster)was most frequent haplogroup in Pakistani and Afghan Hazaras.In MDS plot we have seen that Hazara population from Afghanistan is located closer with Afghan population from Afghanistan and Pathan population from northern Afghanistan which is similar to the results of other study conducted by Haber et al.while Pakistani Hazara lined closer to Kazakh and Mongolian population which is similar to our pervious study‘s results.Conclusions:In conclusion genetic variation in Hazara population and its comparison to several relevant populations were done using various statistical tests.This showed that the Hazara population has close affinity to the Central Asian and Mongol populations.In previous study profile of star-cluster was 10(DYS389I),16(DYS389II),25(DYS390),10(DYS391),11(DYS392),13(DYS393),14(DYS388),12(DYS425),11(DYS426),11(DYS434),11(DYS435),12(DYS436),8(DYS437),10(DYS438),10(DYS439).In current study most frequent haplotype was 15-13-29-24-10-11-13-14-11-12 for loci DYS19-DYS389I-DYS389II-DYS390-DYS391-DYS392-DYS393-DYS437-DYS438-DYS439 found in 43 individuals while 14-13-29-24-8-11-13-14-11-11 found in 9 individuals and 15-13-29-24-11-11-13-14-11-12 found in 8 individuals.These haplotypes are most frequent haplotype in Mongols and Kazakhs.Allelic Frequencies on these 10 Y STRs are almost similar to Kazak population of Kazakhstan Central Asia and somehow similar to Mongol population of Inner Mongolia.Our results contributed towards deciphering the origin of Hazara population and supported the view that they had Kazakh and Mongol origins.The forensic parameter calculations showed high discriminatory power with potential applications for forensic casework.We detected 54 null alleles at the locus DYS448that equated to 13%of null alleles at this locus.This was the highest percentage of DYS448 null alleles in a population to date... |
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