Exploring The Forensic And Genetic Characteristics Of Three Chinese Ethnic Groups By Combining Autosomal InDel,Complete Mitochondrial Genome And Y-Chromosome SNP

Objective:The representative Tibetan in Tibet,Mongolian in Inner Mongolia and Uygur in Xinjiang from the western region of China were selected to investigate the insertion/deletion（In Del）genetic polymorphism in three populations using the Sifa In Del 45plex system,so as to provide population genetic data for forensic identification and forensic genetic research of the system in the three populations.Subsequently,combined with three genetic markers including autosomal insertion/deletion（A-In Del）polymorphism molecular genetic marker,complete mitochondrial genome（mitochondrial DNA,mt DNA）and Y chromosome single nucleotide polymorphism（Y-chromosome SNP,Y-SNP）,the genetic structures and characteristics of the three populations were comprehensively explored from the perspectives of autosome,maternal aspect and paternal.Methods:1.Blood samples were collected from a total of 690 healthy unrelated individuals from three populations,239 from the Tibetan in Tibet,198 from the Mongolian in Inner Mongolia and 253 from the Uygur in Xinjiang.DNA was extracted using the QIAamp DNA Blood Mini kit.2.A total of 690 samples from three populations were subjected to 45 In Del loci complex amplification using the Sifa In Del 45plex system,PCR products were subjected to capillary electrophoresis using the 3130xl gene analyzer,and genotyping by the Gene Mapper ^TMID v3.2.1 software.3.A total of 589 samples from three populations were subjected to mitochondrial whole genome sequencing based on Illumina second-generation sequencing platform.The obtained FASTQ data were imported into haplogrep and mthap websites,and mtphyl software was used to divide haplogroups.4.A total of 381 Y-SNPs in300 samples from three populations were sequenced based on Illumina second-generation sequencing platform,and the haplogroups were manually divided according to 381 Y-SNP typing and by referring to the ISOGG website.5.Microsoft Excel,Arlequin v3.5.2,SPSS25.0and other softwares were used for statistical analysis of forensic genetic parameters of In Del.Based on the A-In Del allele frequency,mt DNA haplogroup and Y-SNP haplogroup data respectively and the published partial intercontinental population genetic data were compared and integrated,and the population genetic structure characteristics were explored using Arlequin v3.5.2,SPSS25.0 and MEGA 11 software.Results:1.Typing data of 45 In Del loci from three populations were obtained for the first time.After Bonferroni correction,the 27 A-In Del sites and 16 X-In Del loci were in Hardy-Weinberg equilibrium.There was no linkage disequilibrium among all loci.The frequencies of deletion alleles at the 27 A-In Del loci in the Sifa In Del 45plex system ranged from 0.1858（rs2307963）to 0.6820（rs3583316,rs66481141）in the studied populations.The frequencies of deletion alleles at the 16 X-In Del loci ranged from 0.1321（rs36208458）to0.9244（rs200177947）.The polymorphic information content at the 27 A-In Del loci ranged from 0.2568（rs2307963）to 0.3750（rs112052995,rs35248926,rs3837647,rs1160953,rs2067373,and rs2307805）,and the polymorphic information content at the 16 X-In Del loci ranged from 0.1301（rs200177947）to 0.3749（rs 199686249）.The cumulative discrimination power（CDP）of the 27 A-In Del loci in Tibetan in Tibet,Mongolian in Inner Mongolia and Uygur in Xinjiang were 0.999999999974567,0.999999999986552,and 0.999999999999988048,respectively.The combined power of exclusion of trios testing(CPE_trio)were0.997641371,0.997986006 and 0.998004996,respectively.The CDP_femaleof the 16 X-In Del loci was 0.999998454,0.999998389,and 0.999998370 in Tibetan in Tibet,Mongolian in Inner Mongolia and Uygur in Xinjiang female,and CDP_malewas 0.999850112,0.999856063,and 0.999846599 in the male population,respectively.2.The heat map of the frequency of missing alleles at the 27 A-In Del sites showed that the Sifa In Del 45plex system had differences in allele frequency among different intercontinental populations.Through the calculation of the genetic distance F_st,the construction of a phylogenetic tree and multidimensional scaling we have found that the genetic distances of Tibetan in Tibet,Mongolian in Inner Mongolia and Uygur in Xinjiang were closer to those of Han in Changzhou and East Asian populations,while the genetic distances of Uyghur in Xinjiang were closer to those of South Asian populations.3.Mt DNA haplogroups data from three ethnic group were obtained.Among Tibetans in Tibet,M’haplogroup had the largest share,at31%,while N*haplogroup and X haplogroup had the smallest share,both at 1%.Among the Mongolians in Inner Mongolia,D haplogroup and R haplogroup had the largest proportion,both accounting for 23.26%,while Y haplogroup had the smallest proportion,accounting for0.39%.Uygur in Xinjiang has the largest proportion of R haplogroup（48%）,and the smallest proportions of W haplogroup and N*haplogroup（1%）.The phylogenetic tree and MDS map were constructed based on the haplogroup frequency data from the 3 study populations and the reference population.The results showed that from the perspective of maternal inheritance,the genetic distances of the three studied populations were closer to those of the populations in East Asia.4.The Y-SNP haplogroup typing results of three ethnic groups were obtained.Among Tibetan in Tibet,D haplogroup had the highest frequency,at 52%,while R haplogroup,Q haplogroup,O1a haplogroup and L haplogroup all had the smallest share,at 1%.Of the Mongolian in Inner Mongolia,C haplogroup had the highest share,at 48%,while Q haplogroup and G haplogroup had the smallest share,both at 1%.Among Uygur in Xinjiang,R haplogroup has the highest proportion of 35%,while I haplogroup and O1b haplogroup have the lowest proportion,both at 1%.The phylogenetic tree and MDS map were constructed based on the haplogroup frequencies of the three studied populations and the reference populations.The results showed that the genetic relationships of the Tibetan in Tibet,Mongolian in Inner Mongolia were similar to those of the East Asian populations,and the genetic distances of the Uygur in Xinjiang and the Kyrgyz in Xinjiang and other populations in Central and South Asia were relatively close.Conclusion:1.The In Del loci contained in the Sifa In Del 45plex system had good genetic polymorphism in all three study populations,and it could meet the requirements of individual identification in forensic genetics or be an effective supplement for parental identification,and could be used to distinguish different intercontinental populations.Based on 27 A-In Del loci,Tibetan in Tibet,Mongolian in Inner Mongolia and Uygur in Xinjiang clustered and had close genetic relationships with East Asian populations.2.The haplogroup of the three studied populations were divided by mt DNA,and it was found that the haplogroups classification and frequencies of the three populations were diverse and uneven,and haplogroups of East Asian origin accounted for a higher proportion of the three populations.Based on mt DNA haplogroup data of different populations,Tibetans in Tibet,Mongolians in Inner Mongolia,Uygur in Xinjiang have closer genetic distances from the populations in East Asia.3.The haplogroups of the Tibetans in Tibet and Mongolians in Inner Mongolia were mostly haplogroups originating from East Asia,and the haplogroups of the Uygur in Xinjiang were mixed from Central Asia and South Asia.Based on the Y-SNP haplogroup data of different populations,Tibetan in Tibet,Mongolian in Inner Mongolia and the populations in East Asia had closer genetic relationships,while Uygur in Xinjiang had closer genetic relationships with the populations in Central Asia and South Asia.4.Combined with A-In Del,mt DNA genome full sequence and Y-SNP analysis,from the autosomal,matrilineal and paternal genetic background,Tibetan in Tibet and Mongolian in Inner Mongolia have closer genetic relationship with the East Asian population,while the Uygur in Xinjiang have a mixed genetic structure of East Asia,Central Asia and South Asia.